Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) represent a major component of the tumor microenvironment and interplay with cancer cells by secreting cytokines, growth factors and extracellular matrix proteins. When estrogen receptor-negative breast cancer MDA-MB-231 cells were treated with the CAF-conditioned medium (CAF-CM), Akt and STAT3 involved in cell proliferation and survival were activated through phosphorylation. CAFs secrete fibroblast growth factor 2 (FGF2), thereby stimulating breast cancer cell progression. Akt activation induced by CAF-CM in MDA-MB-231 cells was abolished when FGF2-neutralizing antibody was added. Treatment of MDA-MB-231 cells directly with FGF2 enhanced the phosphorylation of Akt and the FGF receptor (FGFR) substrate, FRS2α. These events were abrogated by siRNA-mediated silencing of FGFR1. In a xenograft mouse model, co-injection of MDA-MB-231 cells with activated fibroblasts expressing FGF2 dramatically enhanced activation of Akt. Stable knockdown of FGFR1 blunted Akt phosphorylation in xenograft tumors. MDA-MB-231 cells co-cultured with CAFs or directly stimulated with FGF2 exhibited enhanced nuclear localization of FGFR1. Notably, FGF2 stimulation produced reactive oxygen species (ROS) accumulation in MDA-MB-231 cells, and FGF2-induced nuclear accumulation of FGFR1 was abrogated by the ROS scavenging agent, N-acetylcysteine.

A quantitative assay for agarase activity determination using agarose-iodine complex.

Rapid and simple spectrophotometric methods are required to detect various oligosaccharides produced by agar-hydrolysing enzymes. Herein, we present a quantitative agarose-iodine assay for agarase activity determination via the detection of the extent of agarose degradation. The agarose-iodine complex becomes reddish orange upon the addition of Lugol solution, and the enzymatic activity can be detected with ultraviolet-visible spectroscopy at 600 nm. The main advantages of this modified Lugol assay are high sensitivity, simple detection, and cost effectiveness. A novel definition of the unit to measure and compare the activities of agarases is also suggested.

JNK-mediated Ser27 phosphorylation and stabilization of SIRT1 promote growth and progression of colon cancer through deacetylation-dependent activation of Snail.

Sirtuin 1 (SIRT1), an NAD+ -dependent histone/protein deacetylase, has multifaceted functions in various biological events such as inflammation, aging, and energy metabolism. The role of SIRT1 in carcinogenesis, however, is still under debate. Recent studies have indicated that aberrant overexpression of SIRT1 is correlated with metastasis and poor prognosis in several types of malignancy, including colorectal cancer. In the present study, we found that both SIRT1 and SIRT1 phosphorylated on serine 27 were coordinately upregulated in colon cancer patients' tissues and human colon cancer cell lines. This prompted us to investigate a role of phospho-SIRT1 in the context of colon cancer progression. A phosphorylation-defective mutant form of SIRT1, in which serine 27 was substituted by alanine (SIRT1-S27A), exhibited lower protein stability compared to that of wild-type SIRT1. Notably, human colon cancer (HCT-116) cells harboring the SIRT1-S27A mutation showed decreased cell proliferation and reduced capability to form xenograft tumor in athymic nude mice, which was accompanied by diminished transcriptional activity of Snail. HCT-116 cells carrying SIRT1-S27A were less capable of deacetylating the Snail protein, with a concomitant decrease in the levels of interleukin (IL)-6 and IL-8 mRNA transcripts. Taken together, these observations suggest that SIRT1 stabilized through phosphorylation on serine 27 exerts oncogenic effects at least partly through deacetylation-dependent activation of Snail and subsequent transcription of IL-6 and IL-8 in human colon cancer cells.

15-Deoxy-Δ12,14-prostaglandin J2 Induces Epithelial-to-mesenchymal Transition in Human Breast Cancer Cells and Promotes Fibroblast Activation.

In inflammation-associated carcinogenesis, COX-2 is markedly overexpressed, resulting in accumulation of various prostaglandins. 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is one of the terminal products of COX-2-catalyzed arachidonic acid catabolism with oncogenic potential. Epithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells lose their polarity and adhesiveness, and thereby gain migratory and invasive properties. Treatment of human breast cancer MCF-7 cells with 15d-PGJ2 induced EMT as evidenced by increased expression of Snail and ZEB1, with concurrent down-regulation of E-cadherin. Nuclear extract from 15d-PGJ2-treated MCF-7 cells showed the binding of Snail and ZEB1 to E-box sequences present in the E-cadherin promoter, which accounts for repression of E-catherin expression. Unlike 15d-PGJ2, its non-electrophilic analogue 9,10-dihydro-15d-PGJ2 failed to induce EMT, suggesting that the α,ß-unsaturated carbonyl group located in the cyclopentenone ring of 15d-PGJ2 is essential for its oncogenic function. Notably, the mRNA level of interleukin-8 (IL-8)/CXCL8 was highly elevated in 15d-PGJ2-stimulated MCF-7 cells. 15d-PGJ2-induced up-regulation of IL-8/CXCL8 expression was abrogated by silencing of Snail short interfering RNA. Treatment of normal fibroblast with conditioned medium obtained from cultures of MCF-7 cells undergoing EMT induced the expression of activated fibroblast marker proteins, α-smooth muscle actin and fibroblasts activation protein-α. Co-culture of normal fibroblasts with 15d-PGJ2-stimulated MCF-7 cells also activated normal fibroblast cells to cancer associated fibroblasts. Taken together, above findings suggest that 15d-PGJ2 induces EMT through up-regulation of Snail expression and subsequent production of CXCL8 as a putative activator of fibroblasts, which may contribute to tumor-stroma interaction in inflammatory breast cancer microenvironment.

Fibroblast growth factor-2, derived from cancer-associated fibroblasts, stimulates growth and progression of human breast cancer cells via FGFR1 signaling.

Cancer-associated fibroblasts (CAFs) constitute a major compartment of the tumor microenvironment. In the present study, we investigated the role for CAFs in breast cancer progression and underlying molecular mechanisms. Human breast cancer MDA-MB-231 cells treated with the CAF-conditioned media manifested a more proliferative phenotype, as evidenced by enhanced messenger RNA (mRNA) expression of Cyclin D1, c-Myc, and proliferating cell nuclear antigen. Analysis of data from The Cancer Genome Atlas revealed that fibroblast growth factor-2 (FGF2) expression was well correlated with the presence of CAFs. We noticed that the mRNA level of FGF2 in CAFs was higher than that in normal fibroblasts. FGF2 exerts its biological effects through interaction with FGF receptor 1 (FGFR1). In the breast cancer tissue array, 42% estrogen receptor-negative patients coexpressed FGF2 and FGFR1, whereas only 19% estrogen receptor-positive patients exhibited coexpression. CAF-stimulated MDA-MB-231 cell migration and invasiveness were abolished when FGF2-neutralizing antibody was added to the conditioned media of CAFs. In a xenograft mouse model, coinjection of MDA-MB-231 cells with activated fibroblasts expressing FGF2 dramatically enhanced tumor growth, and this was abrogated by silencing of FGFR1 in cancer cells. In addition, treatment of MDA-MB-231 cells with FGF2 enhanced expression of Cyclin D1, a key molecule involved in cell cycle progression. FGF2-induced cell migration and upregulation of Cyclin D1 were abolished by siRNA-mediated FGFR1 silencing. Taken together, the above findings suggest that CAFs promote growth, migration and invasion of MDA-MB-231 cells via the paracrine FGF2-FGFR1 loop in the breast tumor microenvironment.

Genistein Inhibits Proliferation of BRCA1 Mutated Breast Cancer Cells: The GPR30-Akt Axis as a Potential Target.

BACKGROUND: BRCA1 mutated breast cancer cells exhibit the elevated cell proliferation and the higher metastatic potential. G protein-coupled receptor 30 (GPR30) has been shown to regulate growth of hormonally responsive cancers, such as ovarian and breast cancers, and high expression of GPR30 is found in estrogen receptor (ER)-negative breast cancer cells. ER-negative breast cancer patients often have a mutation in the tumor suppressor gene, BRCA1. This study explored antiproliferative effects of genistein, a chemopreventive isoflavone present in legumes, and underlying molecular mechanisms in triple negative breast cancer cells with or without functionally active BRCA1. METHODS: Expression of BRCA1, GPR30 and Nrf2 was measured by Western blot analysis. Reactive oxygen species (ROS) accumulation was monitored by using the fluorescence-generating probe, 2',7'-dichlorofluorescein diacetate. The effects of genistein on breast cancer cell viability and proliferation were assessed by the MTT, migration and clonogenic assays. RESULTS: The expression of GPR30 was dramatically elevated at both transcriptional and translational levels in BRCA1 mutated breast cancer cells compared to cells with wild-type BRCA1. Notably, there was diminished Akt phosporylation in GPR30 silenced cells. Treatment of BRCA1 silenced breast cancer cells with genistein resulted in the down-regulation of GPR30 expression and the inhibition of Akt phosphorylation as well as the reduced cell viability, migration and colony formation. Genistein caused cell cycle arrest at the G2/M phase in BRCA1-mutant cells through down-regulation of cyclin B1 expression. Furthermore, BRCA1-mutant breast cancer cells exhibited higher levels of intracellular ROS than those in the wild-type cells. Genistein treatment lowered the ROS levels through up-regulation of Nrf2 expression. CONCLUSIONS: Lack of functional BRCA1 activates GPR30 signaling, thereby stimulating Akt phosphorylation and cell proliferation. Genistein induces G2/M phase arrest by down-regulating cyclin B1 expression, which is attributable to its suppression of GPR30 activation and Akt phosphorylation in BRCA1 impaired breast cancer cells.

Curcumin suppresses oncogenicity of human colon cancer cells by covalently modifying the cysteine 67 residue of SIRT1.

SIRT1, an NAD+-dependent histone/protein deacetylase, has diverse physiological actions. Recent studies have demonstrated that SIRT1 is overexpressed in colorectal cancer, suggesting its oncogenic potential. However, the molecular mechanisms by which overexpressed SIRT1 induces the progression of colorectal cancer and its inhibition remain largely unknown. Curcumin (diferuloymethane), a major component of the spice turmeric derived from the plant Curcuma longa L., has been reported to exert chemopreventive and anti-carcinogenic effects on colon carcinogenesis. In the present study, we found that curcumin reduced the expression of SIRT1 protein without influencing its mRNA expression in human colon cancer cells, suggesting posttranslational regulation of SIRT1 by this phytochemical. Notably, ubiquitination and subsequent proteasomal degradation of SIRT1 were induced by curcumin treatment. Results of nano-LC-ESI-MS/MS revealed the direct binding of curcumin to cysteine 67 of SIRT1. In line with this result, the protein stability and clonogenicity of a mutant SIRT1 in which cysteine 67 was substituted by alanine were unaffected by curcumin. Taken together, these observations suggest that curcumin facilitates the proteasomal degradation of oncogenic SIRT1 through covalent modification of SIRT1 at the cysteine 67 residue.

15-Deoxy-Δ12,14-prostaglandin J2 activates PI3K-Akt signaling in human breast cancer cells through covalent modification of the tumor suppressor PTEN at cysteine 136.

15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), one of the terminal products of cyclooxygenase-2-catalized arachidonic acid metabolism, has been shown to stimulate breast cancer cell proliferation and migration through Akt activation, but the underlying mechanisms remain poorly understood. In the present study, we investigated the effects of 15d-PGJ2 on the activity of PTEN, the inhibitor of the phosphoinositide 3-kinase (PI3K)-Akt axis, in human breast cancer (MCF-7) cells. Since the α,ß-unsaturated carbonyl moiety in the cyclopentenone ring of 15d-PGJ2 is electrophilic, we hypothesized that 15d-PGJ2-induced Akt phosphorylation might result from the covalent modification and subsequent inactivation of PTEN that has several critical cysteine residues. When treated to MCF-7 cells, 15d-PGJ2 bound to PTEN, and this was abolished in the presence of the thiol-reducing agent dithiothreitol. A mass spectrometric analysis by using recombinant and endogenous PTEN protein revealed that the cysteine 136 residue (Cys136) of PTEN is covalently modified upon treatment with 15d-PGJ2. Notably, the ability of 15d-PGJ2 to covalently bind to PTEN as well as to induce Akt phosphorylation was abolished in the cells expressing a mutant form of PTEN in which Cys136 was replaced by serine (C136S-PTEN). The present study demonstrates for the first time that electrophilic 15d-PGJ2 directly binds to cysteine 136 of PTEN and provides new insight into PTEN loss in cancer progression associated with chronic inflammation. These observations suggest that 15d-PGJ2 can undergo nucleophilic addition to PTEN, presumably at Cys136, thereby inactivating this tumor suppressor protein with concomitant Akt activation.

Resveratrol suppresses migration, invasion and stemness of human breast cancer cells by interfering with tumor-stromal cross-talk.

Cancer-associated fibroblasts (CAFs) constitute a major compartment of the tumor microenvironment. CAFs produce a variety of cytokines, growth factors and extracellular matrix proteins, thereby stimulating tumor progression. CAFs are distinct from normal fibroblasts for their overexpression of α-smooth muscle actin. Recent studies suggest that CAFs play an important role in proliferation and migration of cancer cells through cross-talk with them. Resveratrol (trans-3,4'5,-trihydroxystilbene), a phytoalexin present in grapes, has been reported to possess chemopreventive and chemotherapeutic activities. In the present study, we examined the effects of resveratrol on CAF-induced migration, invasion and self-renewal activity of breast cancer cells. Resveratrol inhibited proliferation, migration and invasion of human breast cancer cells treated with CAF-conditioned media (CAF-CM). Resveratrol treatment suppressed the CAF-CM-induced expression of Cyclin D1, c-Myc, MMP-2 and MMP-9. In addition, resveratrol inhibited Sox2 expression as well as activation of Akt and STAT3 induced by CAF-CM in breast cancer cells. Further, resveratrol abrogated stemness properties and reduced the expression of self-renewal signaling molecules in stem-like breast cancer cells. Taken together, the present study provides insights into the role of resveratrol in tumor microenvironment with focus on interaction between cancer cells and the hosting niche.

Regulation of the tumor suppressor PTEN by natural anticancer compounds.

The tumor suppressor phosphatase and tensin homologue (PTEN) has phosphatase activity, with phosphatidylinositol (3,4,5)-trisphosphate (PIP3), a product of phosphatidylinositol 3-kinase (PI3K), as one of the principal substrates. PTEN is a negative regulator of the Akt pathway, which plays a fundamental role in controlling cell growth, survival, and proliferation. Loss of PTEN function has been observed in many different types of cancer. Functional inactivation of PTEN as a consequence of germ-line mutations or promoter hypermethylation predisposes individuals to malignancies. PTEN undergoes posttranslational modifications, such as oxidation, acetylation, phosphorylation, SUMOylation, and ubiquitination, which influence its catalytic activity, interactions with other proteins, and subcellular localization. Cellular redox status is crucial for posttranslational modification of PTEN and its functional consequences. Oxidative stress and inflammation are major causes of loss of PTEN function. Pharmacologic or nutritional restoration of PTEN function is considered a reliable strategy in the management of PTEN-defective cancer. In this review, we highlight natural compounds, such as curcumin, indol-3 carbinol, and omega-3 fatty acids, that have the potential to restore or potentiate PTEN expression/activity, thereby suppressing cancer cell proliferation, survival, and resistance to chemotherapeutic agents.

15-Deoxy-Δ12,14-prostaglandin J2 stabilizes hypoxia inducible factor-1α through induction of heme oxygenase-1 and direct modification ofprolyl-4-hydroxylase 2.

15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), a representative J-series cyclopentenone prostaglandin, has biphasic roles in cell proliferation and apoptosis. Hypoxia inducible factor-1 (HIF-1) regulates expression of various genes involved in tumor growth and angiogenesis. In the present study, treatment of human breast cancer (MCF-7) cells with 15d-PGJ2 resulted in the accumulation of the α-subunit of HIF-1. Pretreatment with zinc protoporphyrin IX, a pharmacological inhibitor of heme oxygenase-1 (HO-1), as well as siRNA knockdown of HO-1 gene in MCF-7 cells attenuated 15d-PGJ2-mediated HIF-1α accumulation. 15d-PGJ2 treatment increased intracellular production of reactive oxygen species (ROS), which was mediated by HO-1 induction. Preincubation of MCF-7 cells with trolox, a water-soluble form of vitamin E, attenuated 15d-PGJ2-induced HIF-1α expression although HO-1 expression was unchanged. This finding suggests that ROS accumulated as a consequence of HO-1 up-regulation can enhance HIF-1α expression in MCF-7 cells treated with 15d-PGJ2. Alternatively, 15d-PGJ2 was found to covalently bind to HIF-1α prolyl-4-hydroxylase 2 (PHD2) in MCF-7 cells, which hampers the proline hydroxylation of HIF-1α, thereby disrupting ubiquitin-dependent proteasomal degradation of this transcription factor. Pretreatment with thiol reducing agents blunted 15d-PGJ2-induced HIF-1α stabilization, indicative of a cysteine residue as a direct target of 15d-PGJ2. Molecular docking analysis suggests that 15d-PGJ2 preferentially binds to PHD2 in the vicinity of the Cys201 residue based on binding energies and carbon-sulfur distances. In summary, 15d-PGJ2 stabilizes HIF-1α in MCF-7 cells through HO-1 induction with subsequent ROS generation and also through direct modification of PHD2.

Guggulsterone induces heme oxygenase-1 expression through activation of Nrf2 in human mammary epithelial cells: PTEN as a putative target.

Guggulsterone (GS) [4,17(20)-pregnadiene-3,16-dione] is a phytosterol found in the gum resin of the Commiphora mukul. GS exists naturally in two stereoisomers: E-GS (cis-GS) and Z-GS (trans-GS). In this study, the effects of both isomers on expression of the cytoprotective enzyme heme oxygenase-1 (HO-1) were evaluated in human mammary epithelial (MCF10A) cells. NF-E2-related factor 2 (Nrf2) is considered a master regulator in activating antioxidant response element (ARE)-driven expression of HO-1 and many other antioxidant/cytoprotective proteins. cis-GS upregulated the transcription and protein expression of HO-1 to a greater extent than did trans-GS. cis-GS treatment enhanced nuclear translocation and ARE-binding activity of Nrf2. MCF10A cells transfected with an ARE luciferase construct exhibited significantly elevated Nrf2 transcriptional activity upon cis-GS treatment compared with cells transfected with the control vector. In addition, silencing of the Nrf2 gene abrogated cis-GS-induced expression of HO-1. Incubation of MCF10A cells with cis-GS increased phosphorylation of Akt. The pharmacological inhibition of phosphoinositide 3-kinase (PI3K), an upstream kinase responsible for Akt phosphorylation, abrogated cis-GS-induced Nrf2 nuclear translocation. Pretreatment with the thiol-reducing agents attenuated Akt phosphorylation, Nrf2 activation and HO-1 expression, suggesting that cis-GS may cause thiol modification of an upstream signaling modulator. Phosphatase and Tensin Homologue Deleted on Chromosome 10 (PTEN) is a negative regulator of the PI3K-Akt axis. The mutation in cysteine 124 present in the catalytic domain of PTEN abolished cis-GS-induced HO-1 expression as well as Akt phosphorylation. Whether this cysteine is a 'bona fide' target of cis-GS in its activation of Nrf2 needs additional investigation.