Osteosarcoma is the most common primary bone tumor and poor prognosis for osteosarcoma patients is mainly due to chemotherapy resistance. MicroRNAs are important to maintain pathophysiological mechanisms of cancer and influence cell sensitivity to chemotherapy. In this study, we tested the functions of microRNA-21 for malignant features as well as for drug resistance of osteosarcoma. We used Northern blot to measure microRNA-21 levels in osteosarcoma-derived cell lines. MicroRNA-21 activity was modulated by either expressing a sponge to decrease its activity in an osteosarcoma-derived cell line expressing high levels of microRNA-21 or by introducing pri-microRNA-21 in a cell line with low endogenous levels. Cell migration was determined in a scratch assay and cell proliferation was measured by performing growth curve analysis. Sensitivity of the cells towards chemotherapeutics was investigated by performing cell viability assays and calculating the IC50 values. While cell migration was unaffected by modulated microRNA-21 levels, microRNA-21 inhibition slowed proliferation and exogenously expressed microRNA-21 promoted this process. Modulated microRNA-21 activity failed to effect sensitivity of osteosarcoma-derived cell lines to doxorubicin or methotrexate. Contrarily, reduction of microRNA-21 activity resulted in enhanced resistance towards cisplatin while ectopic expression of microRNA-21 showed the opposite effect. Increased microRNA-21 levels repressed the expression of Sprouty2 and ectopic expression of Sprouty2 was able to largely rescue the observed effects of microRNA-21 in osteosarcoma. In summary, our data indicate that in osteosarcoma microRNA-21 expression is an important component for regulation of cell proliferation and for determining sensitivity to cisplatin.

a b s t r a c tAs negative regulators of receptor tyrosine kinase-mediated signalling, Sprouty proteins fulfil important roles during carcinogenesis. In this report, we demonstrate that Sprouty2 protein expression inhibits cell proliferation and migration in osteosarcoma-derived cells. Although earlier reports describe a tumour-promoting function, these results indicate that Sprouty proteins also have the potential to function as tumour suppressors in sarcoma. In contrast to Sprouty2, Sprouty4 expression failed to interfere with proliferation and migration of the osteosarcoma-derived cells, possibly due to a less pronounced interference with mitogen-activated protein kinase activity. Sequences within the NH 2 -terminus are responsible for the specific inhibitory function of Sprouty2 protein.

Sprouty proteins are modulators of mitogen-induced signal transduction processes and therefore can influence the process of cancerogenesis. In particular, Sprouty2 has been shown to have an important role in cancer development of many tumor entities including breast cancer. In this report, we investigated the role of Sprouty4 in breast cancer-derived cell lines. We have found that ectopic Sprouty4 expression inhibits cell proliferation of breast cancer cell lines independently of their endogenous expression levels. Corroborating Sprouty4 downregulation causes accelerated growth. Furthermore, we demonstrate that an increase in Sprouty4 content interferes with serum-induced activation of mitogen-activated protein kinase pathway. Additionally, Sprouty4 expression negatively influences cell migration. These data suggest that Sprouty4 is a possible candidate for a tumor suppressor in breast cancer.

Sprouty1 protein belongs to a family of receptor tyrosine kinase-mediated signaling inhibitors, whose members are usually regulated by growth factors to form a negative feedback loop. Correspondingly fluctuations of Sprouty1 mRNA in response to single growth factors have been observed. In this report, we investigate Sprouty1 protein levels and show that in non-small cell lung carcinoma-derived cells, the expression levels are unaffected by the serum content in the cellular environment. Although cells harboring K-Ras mutations express insignificant higher Sprouty1 levels, ectopic expression of constitutive active Ras in normal human lung fibroblasts fails to augment Sprouty1 protein content. Furthermore, serum starvation for three days has no influence on Sprouty1 expression and addition of serum or of singular growth factors leaves Sprouty protein levels unchanged. Cell cycle analysis reveals that Sprouty1 levels remain constant throughout the whole cell cycle. These data demonstrate that Sprouty1 expression is not connected with mitogenic signaling and cell proliferation.

Abstract. MicroRNAs can govern up to hundred different mRNAs and are important regulators of gene expression programs in development and disease. We analyzed the expression of microRNA-21, one of the most common oncomirs, in non-small cell lung cancer (NSCLC). Using northern blots the microRNA-21 expression levels of NSCLC-derived tissue and cell lines were measured. In line with earlier observations we show that mature microRNA-21 expression levels are highly increased in NSCLC-derived tissue compared to normal lung tissue. Additionally, we demonstrate that microRNA-21 levels correlate with malignancy since its expression in higher staged tumors is significantly more elevated compared to stage 1A. Interestingly, microRNA-21 levels in cultured NSCLC-derived cells are comparable to the expression detected in non-malignant lung tissue. Since microRNA-21 levels showed no fluctuation during the cell cycle, accelerated proliferation of tumor cells is not responsible for microRNA-21 upregulation in the tumor compartment. Similarly to NSCLC-derived cancer cells, the tumor-associated fibroblasts show low expression levels of microRNA-21. Together, these data indicate that rather microenviromental and growth conditional changes than intrinsic features of the cancer cells are responsible for the observed increase of microRNA-21 levels in tumor tissues. Subsequently culturing conditions were changed to assess the impact of co-cultivation with fibroblasts, hypoxia and anchorage-independent growth on microRNA-21 expression. While co-cultivation with tumor-associated fibroblasts had no effect on microRNA-21 expression, both hypoxia and anchorage-independent growth cause a microRNA-21 elevation. In summary, our data demonstrate that growth conditions especially expected in more malignant tumors result in microRNA-21 upregulation explaining the observed increase in higher staged lung cancer tissue, but not in lung cancerderived cells.

MNS16A, a functional polymorphic tandem repeat minisatellite, is located in the promoter region of an antisense transcript of the human telomerase reverse transcriptase gene. MNS16A promoter activity depends on the variable number of tandem repeats (VNTR) presenting varying numbers of transcription factor binding sites for GATA binding protein 1. Although MNS16A has been investigated in multiple cancer epidemiology studies with incongruent findings, functional data of only two VNTRs (VNTR-243 and VNTR-302) were available thus far, linking the shorter VNTR to higher promoter activity.For the first time, we investigated promoter activity of all six VNTRs of MNS16A in cell lines of colorectal, lung and prostate cancer using Luciferase reporter assay. In all investigated cell lines shorter VNTRs showed higher promoter activity. While this anticipated indirect linear relationship was affirmed for colorectal cancer SW480 (P = 0.006), a piecewise linear regression model provided significantly better model fit in lung cancer A-427 (P = 6.9 × 10−9) and prostate cancer LNCaP (P = 0.039). In silico search for transcription factor binding sites in MNS16A core repeat element suggested a higher degree of complexity involving X-box binding protein 1, general transcription factor II–I, and glucocorticoid receptor alpha in addition to GATA binding protein 1.Further functional studies in additional cancers are requested to extend our knowledge of MNS16A functionality uncovering potential cancer type-specific differences. Risk alleles may vary in different malignancies and their determination in vitro could be relevant for interpretation of genotype data.

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