Understanding the influence of substrate when growing tumorspheres.

BACKGROUND: Cancer stem cells are important for the development of many solid tumors. These cells receive promoting and inhibitory signals that depend on the nature of their environment (their niche) and determine cell dynamics. Mechanical stresses are crucial to the initiation and interpretation of these signals. METHODS: A two-population mathematical model of tumorsphere growth is used to interpret the results of a series of experiments recently carried out in Tianjin, China, and extract information about the intraspecific and interspecific interactions between cancer stem cell and differentiated cancer cell populations. RESULTS: The model allows us to reconstruct the time evolution of the cancer stem cell fraction, which was not directly measured. We find that, in the presence of stem cell growth factors, the interspecific cooperation between cancer stem cells and differentiated cancer cells induces a positive feedback loop that determines growth, independently of substrate hardness. In a frustrated attempt to reconstitute the stem cell niche, the number of cancer stem cells increases continuously with a reproduction rate that is enhanced by a hard substrate. For growth on soft agar, intraspecific interactions are always inhibitory, but on hard agar the interactions between stem cells are collaborative while those between differentiated cells are strongly inhibitory. Evidence also suggests that a hard substrate brings about a large fraction of asymmetric stem cell divisions. In the absence of stem cell growth factors, the barrier to differentiation is broken and overall growth is faster, even if the stem cell number is conserved. CONCLUSIONS: Our interpretation of the experimental results validates the centrality of the concept of stem cell niche when tumor growth is fueled by cancer stem cells. Niche memory is found to be responsible for the characteristic population dynamics observed in tumorspheres. The model also shows why substratum stiffness has a deep influence on the behavior of cancer stem cells, stiffer substrates leading to a larger proportion of asymmetric doublings. A specific condition for the growth of the cancer stem cell number is also obtained.

Heregulin Drives Endocrine Resistance by Altering IL-8 Expression in ER-Positive Breast Cancer.

Sustained HER2/HER3 signaling due to the overproduction of the HER3 ligand heregulin (HRG) is proposed as a key contributor to endocrine resistance in estrogen receptor-positive (ER+) breast cancer. The molecular mechanisms linking HER2 transactivation by HRG-bound HER3 to the acquisition of a hormone-independent phenotype in ER+ breast cancer is, however, largely unknown. Here, we explored the possibility that autocrine HRG signaling drives cytokine-related endocrine resistance in ER+ breast cancer cells. We used human cytokine antibody arrays to semi-quantitatively measure the expression level of 60 cytokines and growth factors in the extracellular milieu of MCF-7 cells engineered to overexpress full-length HRGß2 (MCF-7/HRG cells). Interleukin-8 (IL-8), a chemokine closely linked to ER inaction, emerged as one the most differentially expressed cytokines. Cytokine profiling using structural deletion mutants lacking both the N-terminus and the cytoplasmic-transmembrane region of HRGß2-which is not secreted and cannot transactivate HER2-or lacking a nuclear localization signal at the N-terminus-which cannot localize at the nucleus but is actively secreted and transactivates HER2-revealed that the HRG-driven activation of IL-8 expression in ER+ cells required HRG secretion and transactivation of HER2 but not HRG nuclear localization. The functional blockade of IL-8 with a specific antibody inversely regulated ERα-driven transcriptional activation in endocrine-sensitive MCF-7 cells and endocrine-resistant MCF-7/HRG cells. Overall, these findings suggest that IL-8 participates in the HRG-driven endocrine resistance program in ER+/HER2- breast cancer and might illuminate a potential clinical setting for IL8- or CXCR1/2-neutralizing antibodies.

Fatty Acid Synthase Is a Key Enabler for Endocrine Resistance in Heregulin-Overexpressing Luminal B-Like Breast Cancer.

HER2 transactivation by the HER3 ligand heregulin (HRG) promotes an endocrine-resistant phenotype in the estrogen receptor-positive (ER+) luminal-B subtype of breast cancer. The underlying biological mechanisms that link them are, however, incompletely understood. Here, we evaluated the putative role of the lipogenic enzyme fatty acid synthase (FASN) as a major cause of HRG-driven endocrine resistance in ER+/HER2-negative breast cancer cells. MCF-7 cells engineered to stably overexpress HRG (MCF-7/HRG), an in vitro model of tamoxifen/fulvestrant-resistant luminal B-like breast cancer, showed a pronounced up-regulation of FASN gene/FASN protein expression. Autocrine HRG up-regulated FASN expression via HER2 transactivation and downstream activation of PI-3K/AKT and MAPK-ERK1/2 signaling pathways. The HRG-driven FASN-overexpressing phenotype was fully prevented in MCF-7 cells expressing a structural deletion mutant of HRG that is sequestered in a cellular compartment and lacks the ability to promote endocrine-resistance in an autocrine manner. Pharmacological inhibition of FASN activity blocked the estradiol-independent and tamoxifen/fulvestrant-refractory ability of MCF-7/HRG cells to anchorage-independently grow in soft-agar. In vivo treatment with a FASN inhibitor restored the anti-tumor activity of tamoxifen and fulvestrant against fast-growing, hormone-resistant MCF-7/HRG xenograft tumors in mice. Overall, these findings implicate FASN as a key enabler for endocrine resistance in HRG+/HER2- breast cancer and highlight the therapeutic potential of FASN inhibitors for the treatment of endocrine therapy-resistant luminal-B breast cancer.

An olive oil phenolic is a new chemotype of mutant isocitrate dehydrogenase 1 (IDH1) inhibitors.

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene confer an oncogenic gain-of-function activity that allows the conversion of α-ketoglutarate (α-KG) to the oncometabolite R-2-hydroxyglutarate (2HG). The accumulation of 2HG inhibits α-KG-dependent histone and DNA demethylases, thereby generating genome-wide hypermethylation phenotypes with cancer-initiating properties. Several chemotypes of mutant IDH1/2-targeted inhibitors have been reported, and some of them are under evaluation in clinical trials. However, the recognition of acquired resistance to such inhibitors within a few years of clinical use raises an urgent need to discover new mutant IDH1 antagonists. Here, we report that a naturally occurring phenolic compound in extra-virgin olive oil (EVOO) selectively inhibits the production of 2HG by neomorphic IDH1 mutations. In silico docking, molecular dynamics, including steered simulations, predicted the ability of the oleoside decarboxymethyl oleuropein aglycone (DOA) to preferentially occupy the allosteric pocket of mutant IDH1. DOA inhibited the enzymatic activity of recombinant mutant IDH1 (R132H) protein in the low micromolar range, whereas >10-fold higher concentrations were required to inhibit the activity of wild-type (WT) IDH1. DOA suppressed 2HG overproduction in engineered human cells expressing a heterozygous IDH1-R132H mutation. DOA restored the 2HG-suppressed activity of histone demethylases as it fully reversed the hypermethylation of H3K9me3 in IDH1-mutant cells. DOA epigenetically restored the expression of PD-L1, an immunosuppressive gene silenced in IDH1 mutant cells via 2HG-driven DNA hypermethylation. DOA selectively blocked colony formation of IDH1 mutant cells while sparing WT IDH1 isogenic counterparts. In sum, the EVOO-derived oleoside DOA is a new, naturally occurring chemotype of mutant IDH1 inhibitors.

Assessing the distribution of cancer stem cells in tumorspheres.

Cancer Stem Cells presumably drive tumor growth and resistance to conventional cancer treatments. From a previous computational model, we inferred that these cells are not uniformly distributed in the bulk of a tumorsphere. To confirm this result, we cultivated tumorspheres enriched in stem cells, and performed immunofluorescent detection of the stemness marker SOX2 using confocal microscopy. In this article, we present an image processing method that reconstructs the amount and location of the Cancer Stem Cells in the spheroids. Its advantage is the use of a statistical criterion to classify the cells in Stem and Differentiated, instead of setting an arbitrary threshold. Moreover, the analysis of the experimental images presented in this work agrees with the results from our computational models, thus enforcing the notion that the distribution of Cancer Stem Cells in a tumorsphere is non-homogeneous. Additionally, the method presented here provides a useful tool for analyzing any image in which different kinds of cells are stained with different markers.

Post-surgery statin use contributes to favorable outcomes in patients with early breast cancer.

BACKGROUND: Statins are a group of lipid-lowering drugs with pleiotropic effects that include, but are not limited to the inhibition of cholesterol synthesis resulting in a wide range of anti-inflammatory, anti-tumor, immunomodulatory, and anti-thrombotic properties. This study aimed to determine the impact of the prior to- or after- breast surgery usage of statins on the tumor prognosis in breast cancer (BC) patients. METHODS: A cohort of patients diagnosed with early invasive ductal BC (n=301) at the Hospital Italiano de Buenos Aires, Argentina, with a minimum follow-up period of 10 years after the surgical procedure were included and stratified according to the time of use of statins and type of statin used. Then, local relapse-free survival (RFS), metastasis-free survival (MFS), bone metastasis-free survival (BMFS), visceral metastasis-free (VMFS), mixed metastasis (bone and visceral)-free survival (mix-MFS) and overall survival (OS) were analyzed. RESULTS: Statins usage after breast surgery was related with lesser metastatic occurrence (p=0.017), lower number of metastatic foci (p=0.034) and fewer dead events (p=0.041), as well as longer MFS (p=0.013) and OS (p=0.027). When stratified by the nature of statins (hydrophilic or lipophilic), only the relatively hydrophilic statin rosuvastatin (ROSU) had an impact on the increase of MFS and OS (p=0.018 and p=0.030, respectively). CONCLUSION: Post-surgery statins usage was associated with increased MFS and OS, with increased benefits of ROSU over simvastatin (SIM) or atorvastatin (ATOR). These results set the rationale for additional studies addressing the use of statins, and particularly, rosuvastatin, to improve the outcome of BC patients.

Role of hydroxymethylglutharyl-coenzyme A reductase in the induction of stem-like states in breast cancer.

PURPOSE: De novo synthesis of cholesterol and its rate-limiting enzyme, 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMGCR), is deregulated in tumors and critical for tumor cell survival and proliferation. However, the role of HMGCR in the induction and maintenance of stem-like states in tumors remains unclear. METHODS: A compiled public database from breast cancer (BC) patients was analyzed with the web application SurvExpress. Cell Miner was used for the analysis of HMGCR expression and statin sensitivity of the NCI-60 cell lines panel. A CRISPRon system was used to induce HMGCR overexpression in the luminal BC cell line MCF-7 and a lentiviral pLM-OSKM system for the reprogramming of MCF-7 cells. Comparisons were performed by two-tailed unpaired t-test for two groups and one- or two-way ANOVA. RESULTS: Data from BC patients showed that high expression of several members of the cholesterol synthesis pathway were associated with lower recurrence-free survival, particularly in hormone-receptor-positive BC. In silico and in vitro analysis showed that HMGCR is expressed in several BC cancer cell lines, which exhibit a subtype-dependent response to statins in silico and in vitro. A stem-like phenotype was demonstrated upon HMGCR expression in MCF-7 cells, characterized by expression of the pluripotency markers NANOG, SOX2, increased CD44 +/CD24low/ -, CD133 + populations, and increased mammosphere formation ability. Pluripotent and cancer stem cell lines showed high expression of HMGCR, whereas cell reprogramming of MCF-7 cells did not increase HMGCR expression. CONCLUSION: HMGCR induces a stem-like phenotype in BC cells of epithelial nature, thus affecting tumor initiation, progression and statin sensitivity.

Targeting mTOR to overcome resistance to hormone and CDK4/6 inhibitors in ER-positive breast cancer models.

Resistance to therapy remains a major obstacle in cancer management. Although treatment with hormone and CDK4/6 inhibitors is successful in luminal breast cancer, resistance to these treatments is frequent, highlighting the need for novel therapeutic strategies to delay disease progression and improve patient survival. Here, we assessed the mechanisms of acquired resistance using T47D and MCF-7 tamoxifen- and palbociclib-resistant cell-line variants in culture and as xenografts, and patient-derived cells (PDCs) obtained from sensitive or resistant patient-derived xenografts (PDXs). In these models, we analyzed the effect of specific kinase inhibitors on survival, signaling and cellular aggressiveness. Our results revealed that mTOR inhibition is more effective than PI3K inhibition in overcoming resistance, irrespective of PIK3CA mutation status, by decreasing cell proliferation and tumor growth, as well as reducing cell migration and stemness. Moreover, a combination of mTOR and CDK4/6 inhibitors may prevent pathway reactivation downstream of PI3K, interfering with the survival of resistant cells and consequent tumor escape. In conclusion, we highlight the benefits of incorporating mTOR inhibitors into the current therapy in ER + breast cancer. This alternative therapeutic strategy not only enhances the antitumor response but may also delay the emergence of resistance and tumor recurrence.

Binding of the angiogenic/senescence inducer CCN1/CYR61 to integrin α6ß1 drives endocrine resistance in breast cancer cells.

CCN1/CYR61 promotes angiogenesis, tumor growth and chemoresistance by binding to its integrin receptor αvß3 in endothelial and breast cancer (BC) cells. CCN1 controls also tissue regeneration by engaging its integrin receptor α6ß1 to induce fibroblast senescence. Here, we explored if the ability of CCN1 to drive an endocrine resistance phenotype in estrogen receptor-positive BC cells relies on interactions with either αvß3 or α6ß1. First, we took advantage of site-specific mutagenesis abolishing the CCN1 receptor-binding sites to αvß3 and α6ß1 to determine the integrin partner responsible for CCN1-driven endocrine resistance. Second, we explored a putative nuclear role of CCN1 in regulating ERα-driven transcriptional responses. Retroviral forced expression of a CCN1 derivative with a single amino acid change (D125A) that abrogates binding to αvß3 partially phenocopied the endocrine resistance phenotype induced upon overexpression of wild-type (WT) CCN1. Forced expression of the CCN1 mutant TM, which abrogates all the T1, H1, and H2 binding sites to α6ß1, failed to bypass the estrogen requirement for anchorage-independent growth or to promote resistance to tamoxifen. Wild-type CCN1 promoted estradiol-independent transcriptional activity of ERα and enhanced ERα agonist response to tamoxifen. The α6ß1-binding-defective TM-CCN1 mutant lost the ERα co-activator-like behavior of WT-CCN1. Co-immunoprecipitation assays revealed a direct interaction between endogenous CCN1 and ERα, and in vitro approaches confirmed the ability of recombinant CCN1 to bind ERα. CCN1 signaling via α6ß1, but not via αvß3, drives an endocrine resistance phenotype that involves a direct binding of CCN1 to ERα to regulate its transcriptional activity in ER+ BC cells.

Fatty Acid Synthase Confers Tamoxifen Resistance to ER+/HER2+ Breast Cancer.

The identification of clinically important molecular mechanisms driving endocrine resistance is a priority in estrogen receptor-positive (ER+) breast cancer. Although both genomic and non-genomic cross-talk between the ER and growth factor receptors such as human epidermal growth factor receptor 2 (HER2) has frequently been associated with both experimental and clinical endocrine therapy resistance, combined targeting of ER and HER2 has failed to improve overall survival in endocrine non-responsive disease. Herein, we questioned the role of fatty acid synthase (FASN), a lipogenic enzyme linked to HER2-driven breast cancer aggressiveness, in the development and maintenance of hormone-independent growth and resistance to anti-estrogens in ER/HER2-positive (ER+/HER2+) breast cancer. The stimulatory effects of estradiol on FASN gene promoter activity and protein expression were blunted by anti-estrogens in endocrine-responsive breast cancer cells. Conversely, an AKT/MAPK-related constitutive hyperactivation of FASN gene promoter activity was unaltered in response to estradiol in non-endocrine responsive ER+/HER2+ breast cancer cells, and could be further enhanced by tamoxifen. Pharmacological blockade with structurally and mechanistically unrelated FASN inhibitors fully impeded the strong stimulatory activity of tamoxifen on the soft-agar colony forming capacity-an in vitro metric of tumorigenicity-of ER+/HER2+ breast cancer cells. In vivo treatment with a FASN inhibitor completely prevented the agonistic tumor-promoting activity of tamoxifen and fully restored its estrogen antagonist properties against ER/HER2-positive xenograft tumors in mice. Functional cancer proteomic data from The Cancer Proteome Atlas (TCPA) revealed that the ER+/HER2+ subtype was the highest FASN protein expressor compared to basal-like, HER2-enriched, and ER+/HER2-negative breast cancer groups. FASN is a biological determinant of HER2-driven endocrine resistance in ER+ breast cancer. Next-generation, clinical-grade FASN inhibitors may be therapeutically relevant to countering resistance to tamoxifen in FASN-overexpressing ER+/HER2+ breast carcinomas.

Functional blockade of α5ß1 integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells.

BACKGROUND: Cell scattering is a physiological process executed by stem and progenitor cells during embryonic liver development and postnatal organ regeneration. Here, we investigated the genomic events occurring during this process induced by functional blockade of α5ß1 integrin in liver progenitor cells. RESULTS: Cells treated with a specific antibody against α5ß1 integrin exhibited cell spreading and scattering, over-expression of liver stem/progenitor cell markers and activation of the ERK1/2 and p38 MAPKs signaling cascades, in a similar manner to the process triggered by HGF/SF1 stimulation. Gene expression profiling revealed marked transcriptional changes of genes involved in cell adhesion and migration, as well as genes encoding chromatin remodeling factors. These responses were accompanied by conspicuous spatial reorganization of centromeres, while integrin genes conserved their spatial positioning in the interphase nucleus. CONCLUSION: Collectively, our results demonstrate that α5ß1 integrin functional blockade induces cell migration of hepatic progenitor cells, and that this involves a dramatic remodeling of the nuclear landscape.

Spatial link between nucleoli and expression of the Zac1 gene.

Eukaryotic genomes are highly organized within the cell nucleus. Genome organization not only implies the preferential positioning of genetic elements in the interphase nucleus but also the topographic distribution of biological processes. We have investigated the relationship between spatial organization and genome function in single cells. Myc, c-Met, Igf2r, Asb4, and Zac1 genes have the same radial distribution, but they are not positioned in close proximity with respect to each other. Three-dimensional mapping of their transcription sites uncovered a gene-specific pattern of relative positioning with respect to the nucleolus. We found that the Zac1 gene transcription preferentially occurs juxtaposed to the nucleolus, and that its mRNA accumulates at this site of transcription. Nucleoli isolation followed by qRT-PCR provided evidence for a physical interaction between Zac1 mRNA and the nucleolus. Actinomycin-D treatment induced disassembly of the nucleolus, loss of the RNA-FISH signal, and dramatic increase of the ZAC protein level. However, inhibition of RNA polymerase II had no effect over the Zac1 FISH signal and the protein expression. Induction of cell cycle arrest, which involves participation of the ZAC protein, provoked mRNA release from its retention site and protein synthesis. Our data demonstrate that Zac1 mRNA preferentially accumulates in close proximity to nucleoli within the cell nucleus. In addition, our results suggest a functional link between such spatial distribution and protein expression.

Progesterone receptor isoform-dependent cross-talk between prolactin and fatty acid synthase in breast cancer.

Progesterone receptor (PR) isoforms can drive unique phenotypes in luminal breast cancer (BC). Here, we hypothesized that PR-B and PR-A isoforms differentially modify the cross-talk between prolactin and fatty acid synthase (FASN) in BC. We profiled the responsiveness of the FASN gene promoter to prolactin in T47Dco BC cells constitutively expressing PR-A and PR-B, in the PR-null variant T47D-Y cell line, and in PR-null T47D-Y cells engineered to stably re-express PR-A (T47D-YA) or PR-B (T47D-YB). The capacity of prolactin to up-regulate FASN gene promoter activity in T47Dco cells was lost in T47D-Y and TD47-YA cells. Constitutively up-regulated FASN gene expression in T47-YB cells and its further stimulation by prolactin were both suppressed by the prolactin receptor antagonist hPRL-G129R. The ability of the FASN inhibitor C75 to decrease prolactin secretion was more conspicuous in T47-YB cells. In T47D-Y cells, which secreted notably less prolactin and downregulated prolactin receptor expression relative to T47Dco cells, FASN blockade resulted in an augmented secretion of prolactin and up-regulation of prolactin receptor expression. Our data reveal unforeseen PR-B isoform-specific regulatory actions in the cross-talk between prolactin and FASN signaling in BC. These findings might provide new PR-B/FASN-centered predictive and therapeutic modalities in luminal intrinsic BC subtypes.

Clinical and therapeutic relevance of the metabolic oncogene fatty acid synthase in HER2+ breast cancer.

Fatty acid synthase (FASN) is a key lipogenic enzyme for de novo fatty acid biosynthesis and a druggable metabolic oncoprotein that is activated in most human cancers. We evaluated whether the HER2-driven lipogenic phenotype might represent a biomarker for sensitivity to pharmacological FASN blockade. A majority of clinically HER2-positive tumors were scored as FASN overexpressors in a series of almost 200 patients with invasive breast carcinoma. Re-classification of HER2-positive breast tumors based on FASN gene expression predicted a significantly inferior relapse-free and distant metastasis-free survival in HER2+/FASN+ patients. Notably, non-tumorigenic MCF10A breast epithelial cells engineered to overexpress HER2 upregulated FASN gene expression, and the FASN inhibitor C75 abolished HER2-induced anchorage-independent growth and survival. Furthermore, in the presence of high concentrations of C75, HER2-negative MCF-7 breast cancer cells overexpressing HER2 (MCF-7/HER2) had significantly higher levels of apoptosis than HER2-negative cells. Finally, C75 at non-cytotoxic concentrations significantly reduced the capacity of MCF-7/HER2 cells to form mammospheres, an in vitro indicator of cancer stem-like cells. Collectively, our findings strongly suggest that the HER2-FASN lipogenic axis delineates a group of breast cancer patients that might benefit from treatment with therapeutic regimens containing FASN inhibitors.

AlphaVbeta3 integrin regulates heregulin (HRG)-induced cell proliferation and survival in breast cancer.

alpha(v)beta(3) integrin-overexpression in tumor associated vasculature is a marker of poor prognosis in breast cancer. A positive correlation between alpha(v)beta(3) integrin and overexpression of Heregulin (HRG), a growth factor associated with breast cancer aggressiveness was recently demonstrated. Here, we addressed the role of alpha(v)beta(3) in the proliferation and survival of HRG-overexpressing breast cancer models. Expression of the RGD-binding integrins alpha(v)beta(3), alpha(v)beta(5) and alpha(v)beta(6) was assessed in the HRG-overexpressing breast cancer cells MDA-MB-231, Hs578T (231/WT and Hs578T/WT, respectively) and derived cells transfected with the antisense orientation of the HRG-beta2 full-length cDNA (231/ASPOOL, 231/AS31 and Hs578T/AS15). Interestingly, only alpha(v)beta(3) expression was noticeably decreased by blockade of HRG expression in the 231/ASPOOL, 231/AS31 and Hs578T/AS15 cells. Small RGD-based peptidomimetic alpha(v)beta(3) antagonists significantly decreased cell viability and anchorage-dependent cell growth of HRG-overexpressing cells, while the low-HRG-expressing 231/AS31 cells did not show a significant response. Mechanistically, functional blockade of alpha(v)beta(3) impaired HRG-promoted hyperactivation of ERK1/ERK2 MAPK without altering the activation of AKT. Flow cytometric analysis of the cell cycle demonstrated that alpha(v)beta(3) antagonists significantly decreased S- and G2/M-phase subpopulations of 231/WT and control 231/VEC cells. Comparable, this effect was linked to an increase in the levels and nuclear translocation of the CDKs inhibitor p27(Kip1). Besides downregulating alpha(v)beta(3) and its driven signaling, HRG blockade led to decreased levels of CYR61 in 231/ASPOOL and 231/AS31 cells. Considering that CYR61 is sufficient to upregulate the expression of alpha(v)beta(3), we then assessed alpha(v)beta(3) levels in MDA-MB-231 cell derivatives expressing the antisense orientation of the CYR61 cDNA (231/CYR61AS-5 and 231/CYR61AS-8). Remarkably, downregulation of CYR61 drastically decreased the levels of alpha(v)beta(3) in the 231/CYR61-5 and 231/CYR61-8 cells, providing further evidence of a key role for CYR61 in HRG-dependent alpha(v)beta(3) overexpression. Moreover, blockade of CYR61 expression impaired the HRG-induced hyperactivation of ERK1/ERK2 MAPK without altering the activation status of AKT in MDA-MB-231 cells, thus resembling the effects exerted by the downregulation of HRG expression as well as by functional blockade of alpha(v)beta(3). These results indicate that HRG is regulating alpha(v)beta(3) levels as well as alpha(v)beta(3)-triggered signaling through its downstream effector, CYR61, in highly invasive breast cancer cells. Altogether, the data presented here provide evidence of a CYR61-regulatory role on alpha(v)beta(3) integrin expression in the modulation of uncontrolled growth of HRG-overexpressing breast carcinomas. This work supports additional studies concerning the use of integrin antagonists as dual therapeutic agents in breast cancer, targeting both, endothelial and tumor cells.

A novel CYR61-triggered 'CYR61-alphavbeta3 integrin loop' regulates breast cancer cell survival and chemosensitivity through activation of ERK1/ERK2 MAPK signaling pathway.

The angiogenic inducer CYR61 is differentially overexpressed in breast cancer cells exhibiting high levels of Heregulin (HRG), a growth factor closely associated with a metastatic breast cancer phenotype. Here, we examined whether CYR61, independently of HRG, actively regulates breast cancer cell survival and chemosensitivity, and the pathways involved. Forced expression of CYR61 in HRG-negative MCF-7 cells notably upregulated the expression of its own integrin receptor alphavbeta3 (>200 times). Small peptidomimetic alphavbeta3 integrin antagonists dramatically decreased cell viability of CYR61-overexpressing MCF-7 cells, whereas control MCF-7/V remained insensitive. Mechanistically, functional blockade of alphavbeta3 specifically abolished CYR6-induced hyperactivation of ERK1/ERK2 MAPK, whereas the activation status of AKT did not decrease. Moreover, CYR61 overexpression rendered MCF-7 cells significantly resistant (>10-fold) to Taxol-induced cytotoxicity. Remarkably, alphavbeta3 inhibition converted the CYR61-induced Taxol-resistant phenotype into a hypersensitive one. Thus, the augmentation of Taxol-induced apoptotic cell death in the presence of alphavbeta3 antagonists demonstrated a strong synergism as verified by the terminal transferase-mediated dUTP nick-end labeling (TUNEL) assay and by flow cytometric analysis for DNA content. Indeed, functional blockade of alphavbeta3, similarly to the pharmacological MAPK inhibitor U0126, synergistically increased both the proportion of CYR61-overexpressing breast cancer cells in the G2 phase of the cell cycle and the appearance of sub-G1 hypodiploid (apoptotic) cells caused by Taxol. Strikingly, CYR61 overexpression impaired the accumulation of wild-type p53 following Taxol exposure, while inhibition of alphavbeta3 or ERK1/ERK2 MAPK signalings completely restored Taxol-induced upregulation of p53. Moreover, antisense downregulation of CYR61 expression abolished the anchorage-independent growth of breast cancer cells engineered to overexpress HRG, and significantly increased their sensitivity to Taxol. Our data provide evidence that CYR61 is sufficient to promote breast cancer cell proliferation, cell survival, and Taxol resistance through a alphavbeta3-activated ERK1/ERK2 MAPK signaling. The identification of a 'CYR61-alphavbeta3 autocrine loop' in the epithelial compartment of breast carcinoma strongly suggests that targeting alphavbeta3 may simultaneously prevent breast cancer angiogenesis, growth, and chemoresistance.

A genomic explanation connecting "Mediterranean diet", olive oil and cancer: oleic acid, the main monounsaturated fatty acid of olive oil, induces formation of inhibitory "PEA3 transcription factor-PEA3 DNA binding site" complexes at the Her-2/neu (erbB-2) oncogene promoter in breast, ovarian and stomach cancer cells.

Olive oil is an integral ingredient of the "Mediterranean diet" and accumulating evidence suggests that it may have a potential role in lowering risk of several cancers. We recently hypothesized that the anti-cancer actions of olive oil may relate to its monounsaturated fatty acid (MUFA) oleic acid (OA; 18:1n-9) content to specifically regulate oncogenes. In this study, transient transfection experiments with human Her-2/neu promoter-driven luciferase gene established the ability of OA to specifically repress the transcriptional activity of Her-2/neu gene. Gene repression was seen in tumour-derived cell lines with Her-2/neu gene amplification and overexpression, including SK-Br3 (56% reduction), SK-OV3 (75% reduction) and NCI-N87 (55% reduction) breast, ovarian and stomach cancer cell lines, respectively. Also marginal decreases in promoter activity were observed in cancer cells expressing physiological levels of Her-2/neu (20% reduction in MCF-7 breast cancer cells). Remarkably, OA treatment in Her-2/neu-overexpressing cancer cells was found to induce up-regulation of the Ets protein polyomavirus enhancer activator 3 (PEA3), a transcriptional repressor of Her-2/neu promoter. Also, an intact PEA3 DNA-binding-site at endogenous Her-2/neu gene promoter was essential for OA-induced repression of this gene. Moreover, OA treatment failed to decrease Her-2/neu protein levels in MCF-7/Her2-18 transfectants, which stably express full-length human Her-2/neu cDNA controlled by a SV40 viral promoter. OA-induced transcriptional repression of Her-2/neu through the action of PEA3 protein at the promoter level may represent a novel mechanism linking "Mediterranean diet" and cancer.

DNA topoisomerase IIalpha (TOP2A) inhibitors up-regulate fatty acid synthase gene expression in SK-Br3 breast cancer cells: in vitro evidence for a 'functional amplicon' involving FAS, Her-2/neu and TOP2A genes.

Fatty acid synthase (FAS), the key metabolic multi-enzyme that is responsible for the terminal catalytic step in the de novo fatty acid biosynthesis, plays an active role in the development, maintenance, and enhancement of the malignant phenotype in a subset of breast carcinomas. We recently described that a molecular bi-directional cross-talk between FAS and the Her-2/neu (erbB-2) oncogene is taking place at the level of transcription, translation, and activity in breast cancer cells. Because Her-2/neu has been linked with altered sensitivity to cytotoxic drugs, we envisioned that FAS gene expression may represent a novel predictive molecular factor for breast cancer response to chemotherapy in a Her-2/neu-related manner. We herein evaluated whether chemotherapy-induced cell damage acts in an epigenetic fashion by inducing changes in the transcriptional activation of FAS gene in breast cancer cells. To evaluate this option, FAS- and Her-2/neu-overexpressing SK-Br3 breast cancer cells were transiently transfected with a FAS promoter-reporter construct (FAS-Luciferase) harboring all the elements necessary for high level expression in cancer cells. SK-Br3 cells cultured in the presence of topoisomerase IIalpha (TOP2A) inhibitors doxorubicin and etopoxide (VP-16) demonstrated a 2- to 3-fold increase in FAS promoter activity when compared with control cells growing in drug-free culture conditions. We failed to observe any significant activation of FAS promoter following exposure to the anti-metabolite 5-fluorouracil, the alkylating drug cisplatin, or the microtubule interfering-agents paclitaxel and vincristine. Moreover, the up-regulatory effects of TOP2A inhibitors on the transcriptional activation of FAS gene expression were not significantly decreased when the FAS promoter was damaged at the sterol regulatory element binding protein (SREBP)-binding site. Considering that FAS inhibition produces profound inhibition of DNA replication and S-phase progression in cancer cells, we finally asked whether a cross-talk between TOP2A and FAS could exhibit a Her-2/neu-related bi-directional nature. TOP2A protein levels were decreased during treatment with the anti-Her-2/neu antibody trastuzumab while, concomitantly, FAS promoter activity and FAS protein expression were significantly reduced. Of note, when the expression levels of TOP2A protein were analyzed following exposure of SK-Br3 cells to increasing concentrations of the novel slow-binding FAS inhibitor C75, a dose-dependent reduction in TOP2A expression was observed. Although FAS gene is not physically located in the Her-2/neu-TOP2A amplicon, our present findings strongly suggest that a tight functional association between FAS, Her-2/neu and TOP2A genes is taking place in a subset of breast carcinoma cells.

The metastasis inducer CCN1 (CYR61) activates the fatty acid synthase (FASN)-driven lipogenic phenotype in breast cancer cells.

The angiogenic inducer CCN1 (Cysteine-rich 61, CYR61) is differentially activated in metastatic breast carcinomas. However, little is known about the precise mechanisms that underlie the pro-metastatic actions of CCN1. Here, we investigated the impact of CCN1 expression on fatty acid synthase (FASN), a metabolic oncogene thought to provide cancer cells with proliferative and survival advantages. Forced expression of CCN1 in MCF-7 cells robustly up-regulated FASN protein expression and also significantly increased FASN gene promoter activity 2- to 3-fold, whereas deletion of the sterol response element-binding protein (SREBP) binding site in the FASN promoter completely abrogated CCN1-driven transcriptional activation. Pharmacological blockade of MAPK or PI-3'K activation similarly prevented the ability of CCN1 to induce FASN gene activation. Pharmacological inhibition of FASN activity with the mycotoxin cerulenin or the small compound C75 reversed CCN1-induced acquisition of estrogen independence and resistance to hormone therapies such as tamoxifen and fulvestrant in anchorage-independent growth assays. This study uncovers FASNdependent endogenous lipogenesis as a new mechanism controlling the metastatic phenotype promoted by CCN1. Because estrogen independence and progression to a metastatic phenotype are hallmarks of therapeutic resistance and mortality in breast cancer, this previously unrecognized CCN1-driven lipogenic phenotype represents a novel metabolic target to clinically manage metastatic disease progression.

Additive apoptotic effect of STI571 with the cytoprotective agent amifostine in K-562 cell line.

PURPOSE: To study the apoptotic effect of the 2-phenylaminopyrimidine derivative STI571 in combination with antioxidant agents on K-562 cell line derived from a Philadelphia chromosome-positive chronic myeloid leukemia patient. MATERIALS AND METHODS: K-562 (BCR/ABL+), U-937, and HL60 (BCR/ABL-) leukemic cell lines were incubated with STI571 and the antioxidant agents catalase, glutathione, superoxide dismutase, and amifostine (AMI). Apoptotic effect was analyzed by morphological and flow cytometric criteria. RESULTS: STI571 at concentrations higher than 0.25 mumol L(-1) produced apoptosis (P<0.05) in K-562 cells only after treatment for 72 h. At the mentioned concentrations, STI571 also induced an increase in the loss of mitochondrial transmembrane potential from 24.6 to 40%. Combination of STI571 (0.5 micromol L(-1)) with antioxidant agents showed that the cytoprotective agent AMI (0.75 mg mL(-1)) produced an additive effect in the proapoptotic activity of STI571 in K-562 cells at nuclear (58.8%+/-2.0 vs. 28.9%+/-3.3) and mitochondrial (53.3%+/-3.6 vs. 29.5%+/-1.2) levels. CONCLUSIONS: Our results show that only AMI in combination with STI571, at submicromolar concentration, has an additive effect in K-562 cell line, and it does not have severe toxic effects on Philadelphia chromosome negative cells.