Correction: Therapeutic targeting of Id2 reduces growth of human colorectal carcinoma in the murine liver.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Antiangiogenic and tumour inhibitory effects of downregulating tumour endothelial FABP4.

Fatty acid binding protein 4 (FABP4) is a fatty acid chaperone, which is induced during adipocyte differentiation. Previously we have shown that FABP4 in endothelial cells is induced by the NOTCH1 signalling pathway, the latter of which is involved in mechanisms of resistance to antiangiogenic tumour therapy. Here, we investigated the role of FABP4 in endothelial fatty acid metabolism and tumour angiogenesis. We analysed the effect of transient FABP4 knockdown in human umbilical vein endothelial cells on fatty acid metabolism, viability and angiogenesis. Through therapeutic delivery of siRNA targeting mouse FABP4, we investigated the effect of endothelial FABP4 knockdown on tumour growth and blood vessel formation. In vitro, siRNA-mediated FABP4 knockdown in endothelial cells led to a marked increase of endothelial fatty acid oxidation, an increase of reactive oxygen species and decreased angiogenesis. In vivo, we found that increased NOTCH1 signalling in tumour xenografts led to increased expression of endothelial FABP4 that decreased when NOTCH1 and VEGFA inhibitors were used in combination. Angiogenesis, growth and metastasis in ovarian tumour xenografts were markedly inhibited by therapeutic siRNA delivery targeting mouse endothelial FABP4. Therapeutic targeting of endothelial FABP4 by siRNA in vivo has antiangiogenic and antitumour effects with minimal toxicity and should be investigated further.

Hypoxia-upregulated microRNA-630 targets Dicer, leading to increased tumor progression.

MicroRNAs (miRNAs) are small RNA molecules that affect cellular processes by controlling gene expression. Recent studies have shown that hypoxia downregulates Drosha and Dicer, key enzymes in miRNA biogenesis, causing a decreased pool of miRNAs in cancer and resulting in increased tumor growth and metastasis. Here we demonstrate a previously unrecognized mechanism by which hypoxia downregulates Dicer. We found that miR-630, which is upregulated under hypoxic conditions, targets and downregulates Dicer expression. In an orthotopic mouse model of ovarian cancer, delivery of miR-630 using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) nanoliposomes resulted in increased tumor growth and metastasis, and decreased Dicer expression. Treatment with the combination of anti-miR-630 and anti-vascular endothelial growth factor antibody in mice resulted in rescue of Dicer expression and significantly decreased tumor growth and metastasis. These results indicate that targeting miR-630 is a promising approach to overcome Dicer deregulation in cancer. As demonstrated in the study, use of DOPC nanoliposomes for anti-miR delivery serves as a better alternative approach to cell line-based overexpression of sense or antisense miRNAs, while avoiding potential in vitro selection effects. Findings from this study provide a new understanding of miRNA biogenesis downregulation observed under hypoxia and suggest therapeutic avenues to target this dysregulation in cancer.

Therapeutic targeting of Id2 reduces growth of human colorectal carcinoma in the murine liver.

During development inhibitor of DNA-bind-2 (Id2) regulates proliferation and differentiation. Id2 expression has been detected in cancer cells, yet its cellular function and validity as a therapeutic target remains largely unknown. Immunohistochemical analysis of colorectal cancer (CRC) specimens revealed that Id2 was undetectable in normal colonic mucosa, but occurs in 40% of primary tumors and in most CRC liver metastases (P<0.0001). Additionally, Id2 was expressed in all CRC cell lines assayed. CRC cells with reduced Id2 expression demonstrated reduced proliferation. Analysis of CRC cell cycle regulatory proteins showed that reducing Id2 levels reduces cyclin D1 levels and increased p21 levels. Reduction of Id2 expression also enhanced tumor cell apoptosis, increasing levels of the pro-apoptotic protein Bim/Bod, and cleavage of caspase-7 and poly (ADP-ribose) polymerase. In vivo studies show tumors derived from cells with decreased Id2 levels formed smaller tumors with fewer metastases compared with tumors with normal levels (P<0.05). Furthermore, intraperitoneal administration of Id2 small interfering RNA (siRNA) conjugated with the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine decreased tumor burden in mice compared with control treatment (P=0.006). We conclude that Id2 is upregulated in CRC, and is important in promoting cell survival. In vivo targeting of Id2 by siRNA establishes that it is a valid therapeutic target where its expression occurs.

Tissue transglutaminase expression promotes cell attachment, invasion and survival in breast cancer cells.

Distant metastasis is frequently observed in patients with breast cancer and is a major cause of cancer-related deaths in these patients. Currently, very little is known about the mechanisms that underlie the development of the metastatic phenotype in breast cancer cells. We previously found that metastatic breast cancer cells express high levels of tissue transglutaminase (TG2), but established no direct link between TG2 and metastasis. In this study, we hypothesized that TG2 plays a role in conferring the metastatic phenotype to breast cancer cells. The results obtained suggested that increased expression of TG2 in breast cancer cells contributes to their increased survival, invasion and motility. We further found that TG2 protein in a metastatic breast cancer MDA-MB231 cells was present on the cell surface in close association with integrins beta1, beta4 and beta5. Downregulation of endogenous TG2 by small interfering RNA inhibited fibronectin (Fn)-mediated cell attachment, survival and invasion. Conversely, ectopic expression of TG2 augmented invasion of breast cancer cells and attachment to Fn-coated surfaces. We conclude that TG2 expression in breast cancer cells plays an important role in the development of the metastatic phenotype.

Implications of increased tissue transglutaminase (TG2) expression in drug-resistant breast cancer (MCF-7) cells.

The development of resistance to chemotherapeutic drugs is a major obstacle to the successful treatment of breast cancer. Ways to block or overcome this resistance are objects of intense research. We have previously shown that cancer cells selected for resistance against chemotherapeutic drugs or isolated from metastatic tumor sites have high levels of a calcium-dependent protein crosslinking enzyme, tissue transglutaminase (TG2) but no direct link between TG2 and resistance was established. As TG2 can associate with the beta members of the integrin family of proteins, we hypothesized that TG2 promotes cell survival signaling pathways by activating integrins on the surface of these cells. To test this hypothesis, we studied the expression of TG2 and its interaction with various integrins in drug-resistant MCF-7 breast cancer cells. TG2 closely associated with beta1 and beta5 integrins on the surface of drug-resistant MCF-7 (MCF-7/Dox and MCF-7/RT) cells. The incubation of TG2-expressing drug-resistant MCF-7 cells on fibronectin (Fn)-coated surfaces strongly activated focal adhesion kinase, an event that leads to the activation of several downstream signaling pathways and, in turn, can confer apoptosis-resistant phenotype to cancer cells. The role of TG2 in Fn-mediated cell attachment, cell growth, and cell survival functions was further analysed by small interfering RNA (siRNA) approach. Inhibition of TG2 by siRNA-inhibited Fn-mediated cell attachment and cell survival functions in drug-resistant MCF-7 cells. We conclude that the expression of TG2 in breast cancer cells contributes to the development of the drug-resistance phenotype by promoting interaction between integrins and Fn.