Here we report the identification of a previously undescribed glycosyltransferase-like gene, GLTx, and its cellular effect on mammary carcinoma cells. GLTx codes for a novel human protein which is highly conserved in primates. Data obtained from the mRNA expression database Oncomine, demonstrate that GLTx is differentially expressed in a variety of cancers including breast cancer. Western blot analysis of a multiple human tissue blot using a rabbit polyclonal antibody against GLTx demonstrated that GLTx was highly expressed in liver, moderately in kidney, intestine and stomach and undetectable in any other tissues. To functionally characterize GLTx, we attempted to establish stable cell lines with forced expression. However, forced expression of GLTx was completely lethal and resulted in severe cell death in the mammary carcinoma cell lines, MCF-7 and BT549, as well as in any other cell lines tested. Staining of MCF-7 cells transiently transfected with a GLTx expression plasmid by either Hoechst 33258 or fluorescein isothiocyanate-conjugated annexin V and propidium iodide confirmed that the observed cell death was not caused by apoptosis. Further investigation confirmed that GLTx expressing cells exhibited cytoplasmic accumulation of autophagosomes, consistent with the induction of autophagy, as measured by the fluorescently labelled autophagosome marker LC3. In addition we have shown that GLTx downregulated BCL-2, an inhibitor of autophagy, as measured by BCL-2 gene promoter luciferase activity assay and western blot assay following transient transfection of a GLTx expressing plasmid. Thus, we have demonstrated that GLTx is a novel autophagic inducer that causes cell death by down regulating BCL-2. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-04-02.

Breast cancer is still the most common malignancy and the leading cause of cancer deaths in both developed and under developed countries. Molecular targeted therapy, in particular monoclonal antibody-based treatment, has emerged as a promising approach to treat this disease because of its high specificity and reduced toxicity. As a consequence, identification of novel molecular therapeutic targets is of crucial importance. In an effort to discover new targets for monoclonal antibody-based targeted therapy, we have identified a novel and secreted hominoid-specific molecule, SHON (secreted hominoid-specific oncogene), in the human mammary carcinoma cell line, MCF-7. Like other hominoid-specific genes, SHON has no known orthologs outside of the primate lineage. It is highly expressed in all cancer cell lines tested so far including breast, lung, liver, stomach, colon and prostate cancer as determined at the mRNA level by RT-PCR and at the protein level by western blotting. Forced expression of SHON in MCF-7 cells significantly increased cell proliferation and survival as demonstrated by 5-bromo-2'-deoxyuridine (BrdU) incorporation and Annexin V/propidium iodide apoptosis assays, respectively. Forced expression of SHON also promoted MCF-7 cell anchorage independent growth in soft agar and enhanced cell migration and invasion. Moreover, forced expression of SHON in MCF-7 cells increased tumour volume in a xenograft model of human breast cancer in immunodeficient mice. Furthermore, depletion of endogenous SHON expression using small interfering RNAs, or functional inhibition using an inhibitory rabbit anti-SHON polyclonal antibody, decreased MCF-7 cell proliferation and survival, and reduced MCF-7 oncogenicity and invasiveness. Therefore, SHON is a novel oncogene for mammary carcinoma cells which may be useful as a therapeutic target for the treatment of breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-03.

Introduction: MicroRNAs (miRNAs) are a class of small non-protein coding RNAs approximately 20–24 nucleotides in length which regulate the expression of genes by pairing with the 3’ untranslated regions of target mRNA molecules and inhibiting translation or promoting mRNA degradation. Mature miRNAs are processed from longer transcripts by the miRNA biogenesis machinery, which includes key enzymes Dicer, Drosha and AGO-2. Altered miRNA expression has been implicated in many pathologies, including breast cancer. In general it is the deregulated expression of individual miRNAs that has been implicated in breast cancer pathogenesis. However, global downregulation of all or the majority of miRNAs has also been observed and can lead to a more invasive phenotype and increased proliferation in various cancers. Global miRNA downregulation has been associated with changes in expression levels of proteins involved in miRNA biogenesis. The growth hormone/insulin-like growth factor-1 axis is emerging as an important mediator of tumour development. Studies investigating hGH expression in human breast cancer have demonstrated that hGH expression is positively correlated with lymph node metastasis, tumour stage, HER-2 status and proliferative index. In mammary carcinoma cells autocrine hGH promotes cell proliferation, survival, migration/invasion and epithelial-to-mesenchymal transition as well as tumour formation in a xenograft model. In the current study we demonstrate that autocrine hGH regulates miRNA biogenesis in breast cancer cells. Methods: Forced expression of hGH was established in the mammary carcinoma cell lines MCF-7 and T47D by stable transfection. miRNA expression was determined using miRNA microarray, quantitative real-time PCR (qPCR) arrays and qPCR assays. The expression levels of genes and proteins involved in miRNA biogenesis were assessed using real-time qPCR assays and Western blotting. Results: More than 90% of miRNAs assayed were downregulated in hGH-transfected MCF-7 and T47D cells, compared to control transfected cells, as demonstrated by miRNA microarray and qPCR array. Changes in miRNA expression determined by microarray and qPCR array were verified using miRNA-specific qPCR assays. In MCF-7 cells, autocrine hGH did not significantly affect the mRNA expression levels of miRNA machinery components Drosha, DGCR8, PACT, TARBP, EXP-5, Dicer and AGO2 when compared to control transfected cells. Whereas in T47D cells autocrine hGH increased mRNA expression of TARBP, PACT and EXP-5 by 2.0, 2.3 and 1.8 fold respectively, compared to control transfected cells. Western blot analysis demonstrated that, autocrine hGH decreased protein levels of Dicer in MCF-7 cells, whereas Drosha and AGO2 were unchanged compared to control transfected cells. In T47D cells autocrine hGH decreased protein levels of Dicer, Drosha and AGO2 when compared to control transfected cells. Conclusion: Our findings demonstrate that autocrine hGH stimulates global downregulation of miRNA expression in breast cancer cells. This may be one mechanism whereby autocrine hGH promotes tumour progression. Our results also indicate that autocrine hGH-mediated global downregulation of miRNA expression may occur through regulation of proteins involved in the miRNA biogenesis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-03-06.