Inhibition of EGFR-AKT axis results in the suppression of ovarian tumors in vitro and in preclinical mouse model

Ovarian cancer is the leading cause of cancer related deaths in women. Genetic alterations including overexpression of EGFR play a crucial role in ovarian carcinogenesis. Here we evaluated the effect of phenethyl isothiocyanate (PEITC) in ovarian tumor cells in vitro and in vivo. Oral administration of 12 μmol PEITC resulted in drastically suppressing ovarian tumor growth in a preclinical mouse model. Our in vitro studies demonstrated that PEITC suppress the growth of SKOV-3, OVCAR-3 and TOV-21G human ovarian cancer cells by inducing apoptosis in a concentration-dependent manner. Growth inhibitory effects of PEITC were mediated by inhibition of EGFR and AKT, which are known to be overexpressed in ovarian tumors. PEITC treatment caused significant down regulation of constitutive protein levels as well as phosphorylation of EGFR at Tyr1068 in various ovarian cancer cells. In addition, PEITC treatment drastically reduced the phosphorylation of AKT which is downstream to EGFR and disrupted mTOR signaling. PEITC treatment also inhibited the kinase activity of AKT as observed by the down regulation of p-GSK in OVCAR-3 and TOV-21G cells. AKT overexpression or TGF treatment blocked PEITC induced apoptosis in ovarian cancer cells. These results suggest that PEITC targets EGFR/AKT pathway in our model. In conclusion, our study suggests that PEITC could be used alone or in combination with other therapeutic agents to treat ovarian cancer. © 2012 Loganathan et al

Increased MicroRNA Activity in Human Cancers

MicroRNAs (miRNAs) are small regulatory RNAs that act by blocking the translation and increasing the degradation of target transcripts. MiRNAs play a critical role in many biological processes including development and differentiation and many studies have shown that major changes in miRNA levels occur in cancer. Since miRNAs degrade target messages, we used this property to develop a novel computational method aimed at determining the actual biological activity of miRNAs using variations in gene expression. Using the method described here, we quantified miRNA activity in papillary thyroid carcinoma and breast cancer, and found a strong and distinctive signal of increased global miRNA activity, embedded in the pertaining gene expression measurements. Interestingly, we found that in these two cancers, miRNA activity is globally increased, and is associated with a global downregulation of miRNA target genes. This downreguation of miRNA regulated genes is particularly noticeable for genes carrying multiple target sites for miRNAs. Among the miRNA-repressed genes, we found a significant enrichment of known tumor suppressors, thereby suggesting that the increased miRNA activity was indeed tumorigenic

p21-Activated Kinase 1 (Pak1) Phosphorylates BAD Directly at Serine 111 In Vitro and Indirectly through Raf-1 at Serine 112

Cell survival depends on the balance between protective and apoptotic signals. When the balance of signals tips towards apoptosis, cells undergo programmed cell death. This balance has profound implications in diseases including cancer. Oncogenes and tumor suppressors are mutated to promote cell survival during tumor development, and many chemotherapeutic drugs kill tumor cells by stimulating apoptosis. BAD is a pro-apoptotic member of the Bcl-2 family of proteins, which can be phosphorylated on numerous sites to modulate binding to Bcl-2 and 14-3-3 proteins and inhibit its pro-apoptotic activities. One of the critical phosphorylation sites is the serine 112 (S112), which can be phosphorylated by several kinases including Pak1.We mapped the Pak phosphorylation sites by making serine to alanine mutations in BAD and testing them as substrates in in vitro kinase assays. We found that the primary phosphorylation site is not S112 but serine 111 (S111), a site that is sometimes found phosphorylated in vivo. In transfection assays of HEK293T cells, we showed that Pak1 required Raf-1 to stimulate phosphorylation on S112. Mutating either S111 or S112 to alanine enhanced binding to Bcl-2, but the double mutant S111/112A bound better to Bcl-2. Moreover, BAD phosphorylation at S111 was observed in several other cell lines, and treating one of them with the Pak1 inhibitor 2,2'-Dihydroxy-1,1'-dinaphthyldisulfide (IPA-3) reduced phosphorylation primarily at S112 and to a smaller extent at S111, while Raf inhibitors only reduced phosphorylation at S112.Together, these findings demonstrate that Pak1 phosphorylates BAD directly at S111, but phosphorylated S112 through Raf-1. These two sites of BAD serve as redundant regulatory sites for Bcl-2 binding

Diazeniumdiolate Mediated Nitrosative Stress Alters Nitric Oxide Homeostasis through Intracellular Calcium and S-Glutathionylation of Nitric Oxide Synthetase

Background: PABA/NO is a diazeniumdiolate that acts as a direct nitrogen monoxide (NO) donor and is in development as an anticancer drug. Its mechanism of action and effect on cells is not yet fully understood. Methodology/Principal Findings: We used HPLC and mass spectrometry to identify a primary nitroaromatic glutathione metabolite of PABA/NO and used fluorescent assays to characterize drug effects on calcium and NO homeostasis, relating these to endothelial nitric oxide synthase (eNOS) activity. Unexpectedly, the glutathione conjugate was found to be a competitive inhibitor of sarcoplasmic/endoplasmic reticulum Ca 2+-ATPase (SERCA) presumably at the same site as thapsigargin, increasing intracellular Ca 2+ release and causing auto-regulation of eNOS through S-glutathionylation. Conclusions/Significance: The initial direct release of NO after PABA/NO was followed by an eNOS-mediated generation of NO as a consequence of drug-induced increase in Ca 2+ flux and calmodulin (CaM) activation. PABA/NO has a unique dual mechanism of action with direct intracellular NO generation combined with metabolite driven regulation of eNO

Cancer Associated Fibroblasts Promote Tumor Growth and Metastasis by Modulating the Tumor Immune Microenvironment in a 4T1 Murine Breast Cancer Model

BACKGROUND:Local inflammation associated with solid tumors commonly results from factors released by tumor cells and the tumor stroma, and promotes tumor progression. Cancer associated fibroblasts comprise a majority of the cells found in tumor stroma and are appealing targets for cancer therapy. Here, our aim was to determine the efficacy of targeting cancer associated fibroblasts for the treatment of metastatic breast cancer. METHODOLOGY/PRINCIPAL FINDINGS:We demonstrate that cancer associated fibroblasts are key modulators of immune polarization in the tumor microenvironment of a 4T1 murine model of metastatic breast cancer. Elimination of cancer associated fibroblasts in vivo by a DNA vaccine targeted to fibroblast activation protein results in a shift of the immune microenvironment from a Th2 to Th1 polarization. This shift is characterized by increased protein expression of IL-2 and IL-7, suppressed recruitment of tumor-associated macrophages, myeloid derived suppressor cells, T regulatory cells, and decreased tumor angiogenesis and lymphangiogenesis. Additionally, the vaccine improved anti-metastatic effects of doxorubicin chemotherapy and enhanced suppression of IL-6 and IL-4 protein expression while increasing recruitment of dendritic cells and CD8(+) T cells. Treatment with the combination therapy also reduced tumor-associated Vegf, Pdgfc, and GM-CSF mRNA and protein expression. CONCLUSIONS/SIGNIFICANCE:Our findings demonstrate that cancer associated fibroblasts promote tumor growth and metastasis through their role as key modulators of immune polarization in the tumor microenvironment and are valid targets for therapy of metastatic breast cancer

Identification of epidermal Pdx1 expression discloses different roles of Notch1 and Notch2 in murine KrasG12D-induced skin carcinogenesis in vivo

Background The Ras and Notch signaling pathways are frequently activated during development to control many diverse cellular processes and are often dysregulated during tumorigenesis. To study the role of Notch and oncogenic Kras signaling in a progenitor cell population, Pdx1-Cre mice were utilized to generate conditional oncogenic KrasG12D mice with ablation of Notch1 and/or Notch2. Methodology/Principal Findings Surprisingly, mice with activated KrasG12D and Notch1 but not Notch2 ablation developed skin papillomas progressing to squamous cell carcinoma providing evidence for Pdx1 expression in the skin. Immunostaining and lineage tracing experiments indicate that PDX1 is present predominantly in the suprabasal layers of the epidermis and rarely in the basal layer. Further analysis of keratinocytes in vitro revealed differentiation-dependent expression of PDX1 in terminally differentiated keratinocytes. PDX1 expression was also increased during wound healing. Further analysis revealed that loss of Notch1 but not Notch2 is critical for skin tumor development. Reasons for this include distinct Notch expression with Notch1 in all layers and Notch2 in the suprabasal layer as well as distinctive p21 and β-catenin signaling inhibition capabilities. Conclusions/Significance Our results provide strong evidence for epidermal expression of Pdx1 as of yet not identified function. In addition, this finding may be relevant for research using Pdx1-Cre transgenic strains. Additionally, our study confirms distinctive expression and functions of Notch1 and Notch2 in the skin supporting the importance of careful dissection of the contribution of individual Notch receptors

Sulindac Enhances the Killing of Cancer Cells Exposed to Oxidative Stress

BACKGROUND:Sulindac is an FDA-approved non-steroidal anti-inflammatory drug (NSAID) that affects prostaglandin production by inhibiting cyclooxygenases (COX) 1 and 2. Sulindac has also been of interest for more than decade as a chemopreventive for adenomatous colorectal polyps and colon cancer. PRINCIPAL FINDINGS:Pretreatment of human colon and lung cancer cells with sulindac enhances killing by an oxidizing agent such as tert-butyl hydroperoxide (TBHP) or hydrogen peroxide. This effect does not involve cyclooxygenase (COX) inhibition. However, under the conditions used, there is a significant increase in reactive oxygen species (ROS) within the cancer cells and a loss of mitochondrial membrane potential, suggesting that cell death is due to apoptosis, which was confirmed by Tunel assay. In contrast, this enhanced killing was not observed with normal lung or colon cells. SIGNIFICANCE:These results indicate that normal and cancer cells handle oxidative stress in different ways and sulindac can enhance this difference. The combination of sulindac and an oxidizing agent could have therapeutic value

Tetrahydrohyperforin and Octahydrohyperforin Are Two New Potent Inhibitors of Angiogenesis

We have previously shown that hyperforin, a phloroglucinol derivative found in St. John's wort, behaves as a potent anti-angiogenic compound. To identify the reactive group(s) mainly involved in this anti-angiogenic effect, we have investigated the anti-angiogenic properties of a series of stable derivatives obtained by oxidative modification of the natural product. In addition, in the present work we have studied the role of the four carbonyl groups present in hyperforin by investigating the potential of some other chemically stable derivatives.The experimental procedures included the analysis of the effects of treatment of endothelial cells with these compounds in cell growth, cell viability, cell migration and zymographic assays, as well as the tube formation assay on Matrigel. Our study with hyperforin and eight derivatives shows that the enolized beta-dicarbonyl system contained in the structure of hyperforin has a dominant role in its antiangiogenic activity. On the other hand, two of the tested hyperforin derivatives, namely, tetrahydrohyperforin and octahydrohyperforin, behave as potent inhibitors of angiogenesis. Additional characterization of these compounds included a cell specificity study of their effects on cell growth, as well as the in vivo Matrigel plug assay.These observations could be useful for the rational design and chemical synthesis of more effective hyperforin derivatives as anti-angiogenic drugs. Altogether, the results indicate that octahydrohyperforin is a more specific and slightly more potent antiangiogenic compound than hyperforin

Characterization of a Novel Small Molecule Subtype Specific Estrogen-Related Receptor α Antagonist in MCF-7 Breast Cancer Cells

The orphan nuclear receptor estrogen-related receptor alpha (ERRalpha) is a member of the nuclear receptor superfamily. It was identified through a search for genes encoding proteins related to estrogen receptor alpha (ERalpha). An endogenous ligand has not been found. Novel ERRalpha antagonists that are highly specific for binding to the ligand binding domain (LBD) of ERRalpha have been recently reported. Research suggests that ERRalpha may be a novel drug target to treat breast cancer and/or metabolic disorders and this has led to an effort to characterize the mechanisms of action of N-[(2Z)-3-(4,5-dihydro-1,3-thiazol-2-yl)-1,3-thiazolidin-2-yl idene]-5H dibenzo[a,d][7]annulen-5-amine, a novel ERRalpha specific antagonist.We demonstrate this ERRalpha ligand inhibits ERRalpha transcriptional activity in MCF-7 cells by luciferase assay but does not affect mRNA levels measured by real-time RT-PCR. Also, ERalpha (ESR1) mRNA levels were not affected upon treatment with the ERRalpha antagonist, but other ERRalpha (ESRRA) target genes such as pS2 (TFF1), osteopontin (SPP1), and aromatase (CYP19A1) mRNA levels decreased. In vitro, the ERRalpha antagonist prevents the constitutive interaction between ERRalpha and nuclear receptor coactivators. Furthermore, we use Western blots to demonstrate ERRalpha protein degradation via the ubiquitin proteasome pathway is increased by the ERRalpha-subtype specific antagonist. We demonstrate by chromatin immunoprecipitation (ChIP) that the interaction between ACADM, ESRRA, and TFF1 endogenous gene promoters and ERRalpha protein is decreased when cells are treated with the ligand. Knocking-down ERRalpha (shRNA) led to similar genomic effects seen when MCF-7 cells were treated with our ERRalpha antagonist.We report the mechanism of action of a novel ERRalpha specific antagonist that inhibits transcriptional activity of ERRalpha, disrupts the constitutive interaction between ERRalpha and nuclear coactivators, and induces proteasome-dependent ERRalpha protein degradation. Additionally, we confirmed that knocking-down ERRalpha lead to similar genomic effects demonstrated in vitro when treated with the ERRalpha specific antagonist