Survival factor NFIL3 restricts FOXO-induced gene expression in cancer.

Depending on the circumstance, FOXO (Forkhead O) (FOXO1, FOXO3, and FOXO4) transcription factors activate the expression of markedly different sets of genes to produce different phenotypic effects. For example, distinct FOXO-regulated transcriptional programs stimulate cell death or enhance organism life span. To gain insight into how FOXOs select specific genes for regulation, we performed a screen for genes that modify FOXO activation of TRAIL, a death receptor ligand capable of inducing extrinsic apoptosis. We discovered that the bZIP transcriptional repressor NFIL3 (nuclear factor interleukin 3-regulated) hindered FOXO transcription factor access to chromatin at the TRAIL promoter by binding to nearby DNA and recruiting histone deacetylase-2 (HDAC2) to reduce histone acetylation. In the same manner, NFIL3 repressed expression of certain FOXO targets--e.g., FAS, GADD45α (growth arrest and DNA damage-inducible, α), and GADD45ß--but not others. NFIL3, which we found to be overexpressed in different cancers, supported tumor cell survival largely through repression of TRAIL and antagonized hydrogen peroxide-induced cell death. Moreover, its expression in cancer was associated with lower patient survival. Therefore, NFIL3 alters cancer cell behavior and FOXO function by acting on chromatin to restrict the menu of FOXO target genes. Targeting of NFIL3 could be of therapeutic benefit for cancer patients.

Integrated molecular pathway analysis informs a synergistic combination therapy targeting PTEN/PI3K and EGFR pathways for basal-like breast cancer.

BACKGROUND: The basal-like breast cancer (BLBC) subtype is characterized by positive staining for basal mammary epithelial cytokeratin markers, lack of hormone receptor and HER2 expression, and poor prognosis with currently no approved molecularly-targeted therapies. The oncogenic signaling pathways driving basal-like tumorigenesis are not fully elucidated. METHODS: One hundred sixteen unselected breast tumors were subjected to integrated analysis of phosphoinositide 3-kinase (PI3K) pathway related molecular aberrations by immunohistochemistry, mutation analysis, and gene expression profiling. Incidence and relationships between molecular biomarkers were characterized. Findings for select biomarkers were validated in an independent series. Synergistic cell killing in vitro and in vivo tumor therapy was investigated in breast cancer cell lines and mouse xenograft models, respectively. RESULTS: Sixty-four % of cases had an oncogenic alteration to PIK3CA, PTEN, or INPP4B; when including upstream kinases HER2 and EGFR, 75 % of cases had one or more aberration including 97 % of estrogen receptor (ER)-negative tumors. PTEN-loss was significantly associated to stathmin and EGFR overexpression, positivity for the BLBC markers cytokeratin 5/14, and the BLBC molecular subtype by gene expression profiling, informing a potential therapeutic combination targeting these pathways in BLBC. Combination treatment of BLBC cell lines with the EGFR-inhibitor gefitinib plus the PI3K pathway inhibitor LY294002 was synergistic, and correspondingly, in an in vivo BLBC xenograft mouse model, gefitinib plus PI3K-inhibitor PWT-458 was more effective than either monotherapy and caused tumor regression. CONCLUSIONS: Our study emphasizes the importance of PI3K/PTEN pathway activity in ER-negative and basal-like breast cancer and supports the future clinical evaluation of combining EGFR and PI3K pathway inhibitors for the treatment of BLBC.

Metformin and erlotinib synergize to inhibit basal breast cancer.

Basal-like breast cancers (BBCs) are enriched for increased EGFR expression and decreased expression of PTEN. We found that treatment with metformin and erlotinib synergistically induced apoptosis in a subset of BBC cell lines. The drug combination led to enhanced reduction of EGFR, AKT, S6 and 4EBP1 phosphorylation, as well as prevented colony formation and inhibited mammosphere outgrowth. Our data with other compounds suggested that biguanides combined with EGFR inhibitors have the potential to outperform other targeted drug combinations and could be employed in other breast cancer subtypes, as well as other tumor types, with activated EGFR and PI3K signaling. Analysis of BBC cell line alterations led to the hypothesis that loss of PTEN sensitized cells to the drug combination which was confirmed using isogenic cell line models with and without PTEN expression. Combined metformin and erlotinib led to partial regression of PTEN-null and EGFR-amplified xenografted MDA-MB-468 BBC tumors with evidence of significant apoptosis, reduction of EGFR and AKT signaling, and lack of altered plasma insulin levels. Combined treatment also inhibited xenografted PTEN null HCC-70 BBC cells. Measurement of trough plasma drug levels in xenografted mice and a separately performed pharmacokinetics modeling study support possible clinical translation.

Merlin is a negative regulator of human melanoma growth.

Merlin is encoded by the neurofibromatosis type 2 (NF2) gene and is a member of the Band 4.1 protein family. This protein acts as a linker that connects cell surface proteins to the actin cytoskeleton. Defects caused by mutations of the NF2 gene give rise to NF2 disease, which is generally characterized by the formation of bilateral vestibular schwannomas and, to a lesser extent, meningiomas and ependymomas. In addition to these tumor types, NF2 is mutated and/or merlin expression is reduced or lost in numerous non-NF2 associated tumors, including melanoma. However, the role of merlin in human melanoma growth and the mechanism underlying its effect are currently unknown. In the present study, we show that merlin knockdown enhances melanoma cell proliferation, migration, and invasion in vitro and that decreased merlin expression promotes subcutaneous melanoma growth in immunocompromised mice. Concordantly, we find that increased expression of merlin in a metastatic melanoma cell line reduced their in vitro migration and proliferation, and diminished their ability to grow in an anchorage independent manner. Increased merlin expression also inhibits in vivo growth of these melanoma cells. Lastly, we demonstrate that higher merlin levels in human melanoma cells promote the H(2)O(2)-induced activation of MST1/2 Ser/Thr kinases, which are known tumor suppressors in the Hippo signaling pathway. Taken together, these results provide for the first time evidence that merlin negatively regulates human melanoma growth, and that loss of merlin, or impaired merlin function, results in an opposite effect. In addition, we show that increased merlin expression leads to enhanced activation of the MTS1/2 kinases, implying the potential roles of MST1/2 in mediating the anti-melanoma effects of merlin.

Lack of effect of coenzyme q10 on doxorubicin cytotoxicity in breast cancer cell cultures.

UNLABELLED: BACKGROUND/HYPOTHESES: Doxorubicin is a standard adjuvant therapy for early-stage breast cancer, and it significantly improves disease-free and overall survival. However, 3% to 20% of breast cancer patients develop chronic cardiomyopathic changes and congestive heart failure because of doxorubicin therapy. Doxorubicin-induced cardiotoxicity is thought to be due to the increased generation of reactive oxygen species within cardiac myocyte mitochondria. Coenzyme Q10 (CoQ10) is a lipid-soluble antioxidant that may protect against mitochondrial reactive oxygen species and thus prevent doxorubicin-induced cardiotoxicity. Despite the potential benefits of CoQ10 in preventing cardiotoxicity, it is not known if CoQ10 diminishes the antineoplastic effects of doxorubicin therapy. STUDY DESIGN: In vitro cell culture experiments. METHODS: Breast cancer cell lines (MDA-MB-468 and BT549) were tested for their ability to uptake exogenous CoQ10 using high-performance liquid chromatography. Breast cancer cell lines were then treated with doxorubicin and a range of CoQ10 concentrations to determine the effect of CoQ10 on doxorubicin's cytotoxicity. RESULTS: This study demonstrated that intracellular and mitochondrial CoQ10 concentrations increased substantially as higher exogenous concentrations were administered to breast cancer cells. CoQ10 had no effect on the ability of doxorubicin to induce apoptosis or inhibit growth or colony formation in both the cell lines tested when applied over a wide dose range, which encompassed typical basal plasma levels and plasma levels above those typically achieved by supplemented patients. CONCLUSION: The clinical testing of CoQ10 as a supplement to prevent doxorubicin-induced cardiotoxicity requires confidence that it does not decrease the efficacy of chemotherapy. These results support the hypothesis that CoQ10 does not alter the antineoplastic properties of doxorubicin. Further in vivo studies, as well as combination chemotherapy studies, would be reassuring before a large-scale clinical testing of CoQ10 as a cardioprotective drug.

Merlin is a potent inhibitor of glioma growth.

Neurofibromatosis 2 (NF2) is an inherited cancer syndrome in which affected individuals develop nervous system tumors, including schwannomas, meningiomas, and ependymomas. The NF2 protein merlin (or schwannomin) is a member of the Band 4.1 superfamily of proteins, which serve as linkers between transmembrane proteins and the actin cytoskeleton. In addition to mutational inactivation of the NF2 gene in NF2-associated tumors, mutations and loss of merlin expression have also been reported in other types of cancers. In the present study, we show that merlin expression is dramatically reduced in human malignant gliomas and that reexpression of functional merlin dramatically inhibits both subcutaneous and intracranial growth of human glioma cells in mice. We further show that merlin reexpression inhibits glioma cell proliferation and promotes apoptosis in vivo. Using microarray analysis, we identify altered expression of specific molecules that play key roles in cell proliferation, survival, and motility. These merlin-induced changes of gene expression were confirmed by real-time quantitative PCR, Western blotting, and functional assays. These results indicate that reexpression of merlin correlates with activation of mammalian sterile 20-like 1/2-large tumor suppressor 2 signaling pathway and inhibition of canonical and noncanonical Wnt signals. Collectively, our results show that merlin is a potent inhibitor of high-grade human glioma.

Overexpression of lysyl oxidase to increase matrix crosslinking and improve tissue strength in dermal wound healing.

In this study, we aimed to increase crosslinking in collagen and elastin in the extracellular matrix through overexpression of lysyl oxidase (LO) in order to improve mechanical strength in dermal wounds during healing. We had used a gene activated matrix (GAM) approach to locally deliver plasmid DNA (pDNA) complexed with polyethylenimine (PEI) in collagen gels at the wound site for localized and sustained transfection of cells involved in the healing process. We first demonstrated in vitro that PEI-pDNA complexes in collagen gels could be taken up and expressed by cultured fibroblasts for at least 20 days. In vitro studies showed that fibroblast-seeded GAMs with the LO transgene exhibited over a 3-fold increase in mechanical strength as compared with a green fluorescent protein (GFP)-transgene control. Addition of an inhibitor of LO abolished this increase. We applied this system in a rat dermal wound healing model and showed that treatment with LO-producing GAMs led to significantly enhanced mechanical strength of the wound site.