Activation of AKT signaling promotes epithelial-mesenchymal transition and tumor growth in colorectal cancer cells.

Activation of the serine-threonine protein kinase AKT has emerged as a central feature of epithelial-mesenchymal transition (EMT), which is the initial step for metastasis in many cancer models, including colorectal cancer. The focus of our study was to dissect the role of AKT and its molecular regulation of EMT in colorectal cancer. HCT-116 colorectal cancer cells stably overexpressing AKT (AKT/HCT-116) showed significantly higher cell proliferation compared with vector-transfected cells (pCMV/HCT-116). Elevated expression of important EMT-related transcription factors and genes such as Snail, Slug, ß-catenin, vimentin, and MMP-9 correlated with increased migration and invasion by AKT/HCT-116 cells. Further, in vivo studies confirmed that AKT/HCT-116 xenografts were highly aggressive and angiogenic in nature compared with pCMV/HCT-116 xenografts. Molecular analysis of tumor samples revealed transcriptional regulation of Snail, Slug, ß-catenin, MMP-2, and MMP-9 in AKT/HCT-116 tumors. These results were supported by immunohistochemistry analysis. Low levels of E-cadherin expression with a concomitant increase in and nuclear localization of ß-catenin were evident in AKT/HCT-116 tumors compared with control tumors. Increased microvessel formation coincident with high expression of Factor VIII and increased numbers of reticulocytes confirmed the angiogenic property of AKT/HCT-116 tumors. Our results confirm the potential role of AKT signaling in regulating EMT and angiogenesis in colorectal cancer and suggest that inhibition of AKT can serve as an important therapeutic strategy in modulating EMT in colorectal cancer growth and metastasis.

A genomics approach to identify susceptibilities of breast cancer cells to "fever-range" hyperthermia.

BACKGROUND: Preclinical and clinical studies have shown for decades that tumor cells demonstrate significantly enhanced sensitivity to "fever range" hyperthermia (increasing the intratumoral temperature to 42-45°C) than normal cells, although it is unknown why cancer cells exhibit this distinctive susceptibility. METHODS: To address this issue, mammary epithelial cells and three malignant breast cancer lines were subjected to hyperthermic shock and microarray, bioinformatics, and network analysis of the global transcription changes was subsequently performed. RESULTS: Bioinformatics analysis differentiated the gene expression patterns that distinguish the heat shock response of normal cells from malignant breast cancer cells, revealing that the gene expression profiles of mammary epithelial cells are completely distinct from malignant breast cancer lines following this treatment. Using gene network analysis, we identified altered expression of transcripts involved in mitotic regulators, histones, and non-protein coding RNAs as the significant processes that differed between the hyperthermic response of mammary epithelial cells and breast cancer cells. We confirmed our data via qPCR and flow cytometric analysis to demonstrate that hyperthermia specifically disrupts the expression of key mitotic regulators and G2/M phase progression in the breast cancer cells. CONCLUSION: These data have identified molecular mechanisms by which breast cancer lines may exhibit enhanced susceptibility to hyperthermic shock.

The regulation and role of osteopontin in malignant transformation and cancer.

Osteopontin (OPN) is a predominantly secreted extracellular matrix glycophosphoprotein which binds to alpha v-containing integrins and has an important role in malignant cell attachment and invasion. High OPN expression in the primary tumor is associated with early metastasis and poor outcome in human breast and other cancers. Forced OPN overexpression in benign cells may induce neoplastic-like cell behaviour including increased attachment and invasion in vitro as well as the ability to metastasize in vivo. Conversely, OPN inhibition by antisense cDNA impedes cell growth and tumor forming capacity. OPN is not mutationally activated in cancer but its expression is regulated by Wnt/Tcf signaling, steroid receptors, growth factors, ras, Ets and AP-1 transcription factors. Presumably these factors are implicated in induction of OPN overexpression in cancer. Greater understanding of the role of OPN in neoplastic change and its transcriptional regulation may enable development of novel cancer treatment strategies.

Pathogenic and therapeutic role of miRNAs in breast cancer.

MicroRNAs (miRNAs) are small noncoding RNA molecules present in all cell types, with sizes that vary from 18 to 28 nucleotides. miRNAs play significant roles in several biological processes, including development, differentiation, metabolism, initiation, and progression of cancer. In recent years, considerable research has been directed towards identifying miRNAs in peripheral blood from circulating tumor cells and disseminating tumor cells. Because these circulatory miRNAs are very stable and reproducible, their identification could be useful as prognostic markers as well as therapeutic agents for many cancers such as breast cancer. In this article, we review the role of specific circulatory miRNAs in breast cancer, with particular emphasis on their clinical importance.

Preclinical safety and activity of recombinant VSV-IFN-ß in an immunocompetent model of squamous cell carcinoma of the head and neck.

BACKGROUND: Recombinant vesicular stomatitis virus expressing interferon-ß (VSV-IFN-ß) has demonstrated antitumor activity in vitro and in vivo. In preparation for clinical testing in human squamous cell carcinoma (SCC) of the head and neck, we conducted preclinical studies of VSV-IFN-ß in syngeneic SCC models. METHODS: In vitro, VSV-IFN-ß (expressing rat or mouse interferon [IFN]-ß)-induced cytotoxicity and propagated in rat (FAT-7) or mouse (SCC-VII) SCC cells during normoxia and hypoxia. In vivo, intratumoral administration of VSV-rat-IFN-ß or VSV-human-IFN-ß in FAT-7 bearing or non-tumor bearing immunocompetent rats did not result in acute organ toxicity or death. RESULTS: VSV-r-IFN-ß replicated predominantly in tumors and a dose dependent anti-VSV antibody response was observed. Intratumoral or intravenous administration of VSV-IFN-ß resulted in growth delay and improved survival compared with controls. CONCLUSION: The above data confirm safety and feasibility of VSV-IFN-ß administration in immunocompetent animals and support its clinical evaluation in advanced human head and neck cancer.

Aberrations in Angiogenic Signaling and MYC Amplifications are Distinguishing Features of Angiosarcoma.

Angiosarcomas are very aggressive, rare malignant tumors that originate from vascular or lymphatic vessels and primarily occur following chemical exposure or radiation therapy. Tumor response to either chemotherapy, radiation, or novel anti-angiogenic therapeutics is very low, and because little is known regarding the aberrant signaling that controls these tumors, personalized treatment options for many of these patients are lacking. In this review, we summarize several recent findings regarding the genomics of angiosarcomas, including new discoveries regarding aberrant angiogenic signaling and Myc amplification as key features of this tumor type.

Rho kinase proteins regulate global miRNA expression in endothelial cells.

BACKGROUND: Therapeutic targeting of Rho-Associated, Coiled-Coil Containing Protein Kinase (ROCK) signaling for tumor cells and tumor endothelium has shown efficacy in pre-clinical tumors models, and a better understanding of how proteins regulate tumor progression will strengthen our knowledge over disease etiology and treatment of patients with cancer. Recent reports have shown that ROCK activity is critical for the expression of a large number of mRNA transcripts across multiple cell types including endothelial cells. MATERIALS AND METHODS: To examine the effects of ROCK proteins on microRNA (miRNA) expression in tumor-forming endothelial cells, we utilized microarrays to evaluate expression levels of 1088 miRNAs in vascular tumor-forming endothelial cells knocked-down for ROCK1 or ROCK2 or treated with a pharmacological inhibitor of ROCK activity. RESULTS: Microarray analysis demonstrated that inhibiting ROCK activity altered global miRNA expression. We confirmed our findings using qPCR and identified cell-cycle progression, calcium transport, and neurogenesis/synaptogenesis as the most highly overrepresented predicted target gene networks for the identified miRNAs whose expression was altered by ROCK inhibition. CONCLUSION: ROCK signaling induces large-scale changes in global miRNA expression and may lead to a better understanding of how these proteins affect aberrant vascular states.

Role of plasminogen activator inhibitor-1 in urokinase's paradoxical in vivo tumor suppressing or promoting effects.

Tumor proteases and inhibitors have been associated with paradoxical effects on tumor progression in preclinical and clinical settings. We previously reported that urokinase (uPA) overexpression delays tumor progression in mammary cancer. This study aimed to determine the role of plasminogen activator inhibitor-1 (PAI-1) on uPA's paradoxical in vivo effects. Using syngeneic murine models, we found that stable uPA overexpression promoted in vivo growth of colon tumors (MC-38) naturally expressing high PAI-1, whereas growth inhibition was observed in renal tumors (RENCA) expressing lower PAI-1 levels. In murine mammary carcinoma (4T1), uPA overexpression shifted the uPA/PAI-1 balance in favor of the protease, resulting in significantly reduced tumor growth and metastases in vivo. Conversely, increased tumor progression was observed in stable PAI-1 overexpressing 4T1 tumors as compared with uPA-overexpressing and control tumors. These effects were associated with downregulation of metastases promoting genes in uPA-overexpressing tumors, such as metalloproteinases, CXCL-1, c-Fos, integrin α-5, VEGF-A, PDGF-α, and IL-1ß. In PAI-1-overexpressing tumors, many of the above genes were upregulated. PAI-1 overexpressing tumors had increased total and new tumor microvessels, and increased tumor cell proliferation, whereas the opposite effects were found in uPA-overexpressing tumors. Finally, PAI-1 downregulation led to significant inhibition of 4T1 tumor growth and metastases in vivo. In conclusion, uPA's dual effects on tumor progression occur in the context of its interactions with endogenous PAI-1 expression. Our studies uncover novel mechanisms of in vivo tumor control by modulation of the balance between tumor proteases and inhibitors, which may be exploited therapeutically.

Identification of genes differentially expressed between benign and osteopontin transformed rat mammary epithelial cells.

BACKGROUND: Osteopontin is a secreted, integrin-binding and phosphorylated acidic glycoprotein which has an important role in tumor progression. FINDINGS: In this study, we have utilized suppressive subtractive hybridization (SSH) to evaluate OPN regulated gene expression, using the Rama 37 benign non-invasive rat mammary cell line and a subclone, Rama 37-OPN. Rama 37-OPN was produced by stably transfecting Rama 37 with an OPN expression vector and it demonstrates increased malignant properties in vitro. Sequence and expression array analysis of the respective cDNA libraries of over 1600 subtracted cDNA fragments revealed 982 ESTs, 45 novel sequences and 659 known genes. The known up-regulated genes in the Rama 37-OPN library code for proteins with a variety of functions including those involved in metabolism, cell adhesion and migration, signal transduction and in apoptosis. Four of the most differentially expressed genes between the benign and in vitro malignant rat mammary cell lines are tumor protein translationally controlled I (TPTI), aryl hydrocarbon receptor nuclear translocator (ARNT), ataxia telangiectasia mutated (ATM) and RAN GTPase (RAN). The largest difference (ca 10,000 fold) between the less aggressively (MCF-7, ZR-75) and more aggressively malignant (MDA MB 231, MDA MB 435S) human breast cancer cell lines is that due to RAN, the next is that due to osteopontin itself. CONCLUSION: The results suggest that enhanced properties associated with the malignant state in vitro induced by osteopontin may be due to, in part, overexpression of RAN GTPase and these biological results are the subject of a subsequent publication 1.