No abstract

Although recent advances in breast cancer treatment regimes have improved patient prognosis, resistance to breast cancer therapies, such as radiotherapy, is still a major clinical challenge. In the current study, we have investigated the role of autocrine human GH (hGH) in resistance to ionising radiation (IR)-based therapy. Cell viability and total cell number assays demonstrated that autocrine hGH promoted cell regrowth in the mammary carcinoma cell lines, MDA-MB-435S and T47D, and the endometrial carcinoma cell line, RL95-2, following treatment with IR. In addition, autocrine hGH enhanced MDA-MB-435S and T47D cell clonogenic survival following radiation exposure. The enhanced clonogenic survival afforded by autocrine hGH was mediated by JAK2 and Src kinases. Investigation into the DNA repair capacity demonstrated that autocrine hGH reduced IR-induced DNA damage in MDA-MB-435S and T47D cells. Functional antagonism of hGH increased RL95-2 sensitivity to IR in cell viability and total cell number assays, reduced clonogenic survival and enhanced the induction of DNA damage. Thus, autocrine hGH reduced sensitivity to treatment with IR in mammary and endometrial carcinoma cell lines in vitro, while functional antagonism of hGH sensitised endometrial carcinoma cells to IR. Functional antagonism of hGH, used in conjunction with radiotherapy, may therefore enhance treatment efficacy and improve the prognosis of patients with breast and endometrial cancer.

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.