Reactivation of SYK expression by inhibition of DNA methylation suppresses breast cancer cell invasiveness

Abstract

The gene product of spleen tyrosine kinase (SYK) has been implicated in the suppression of breast cancer invasion. We previously reported that SYK expression is lost in a subset of breast cancer; primarily by methylation-mediated gene silencing. In our study, we explored the possibility of using a DNA methyltransferase inhibitor, 5-aza-2 -deoxycytidine (AZA), to suppress breast cancer cell invasion by restoring SYK expression. We found that AZA treatment reestablished the expression of SYK(L) that was accompanied by suppression of the invasion capacity of SYK-negative cells. This invasion inhibition was not due to global cellular toxicity since this treatment did not affect overall cell proliferation. This decreased invasiveness by AZA treatment was diminished by piceatannol, a SYK inhibitor, suggesting that SYK play a significant role in AZA-inducible invasion suppression. SYK promoter hypermethylation was found infrequent in pathologically normal mammary tissues or benign lesions (<5%). In contrast, SYK methylation was frequently identified in ductal carcinoma in situ (ϳ45%) and invasive ductal carcinoma (47% in node-negative and 40% in node-positive cases), indicating that the hypermethylation of SYK occurs at a stage prior to the development of invasion phenotypes. All these results suggested a potential use of SYK methylation as a valuable biomarker to detect early cancerous lesions and support the use of AZA as a new reagent to the management of advanced breast cancer. © 2004 Wiley-Liss, Inc. Key words: DNA methylation; breast cancer; spleen tyrosine kinase; invasionThe pathologic features of breast cancer follow a sequential progression through benign proliferative hyperplasia, to hyperplasia with atypia, to carcinoma in situ and eventually to invasive and metastatic disease. 1,2 The clinical manifestation of breast cancer is the progression of disease to stages characterized by local invasion of the auxiliary lymph nodes, followed by metastasis primarily to the lung, bone and liver. Invasion and metastasis are two closely related steps that involve changes in the physical coupling of cells to their microenvironment such as alterations of E-cadherin or ␤-catenin and activation of extracellular proteases. 3,4 The development of local invasion and distant metastasis are strongly associated with low therapeutic efficacy and poor survival. 5,6 The clinical value is thus evident to develop diagnostic biomarkers prior to the establishment of invasive disease and to discover effective treatments.Because of the ineffectiveness of traditional therapeutic measures for breast cancer at its advanced stage, the search for compounds against cancer invasion has long been an intensive research area. Despite continued efforts, no effective measure has been discovered to prevent or treat this type of invasive disease. Targeting some of the most obvious molecules, such as metalloproteinase, has proved ineffective, 7 probably because the metalloproteinase family comprises a large number of highly homologous molecules. Oth...

Cytotoxic activity of gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukemia correlates with the expression of protein kinase Syk

“…33 Moreover, it was demonstrated that loss of Syk expression in breast cancer and T-cell acute lymphoblastic leukemia cells occurs at the transcriptional level, and is a result of DNA hypermethylation. 31,36,37 Treatment of these cells with 5-azacytidine (5-aza), a methylation inhibitor, restored Syk expression and function. 36,37 5-Aza is a nonspecific Syk DNA methyltransferase inhibitor that is used for the treatment of myelodysplasia 38 and possibly AML.…”

“…31,36,37 Treatment of these cells with 5-azacytidine (5-aza), a methylation inhibitor, restored Syk expression and function. 36,37 5-Aza is a nonspecific Syk DNA methyltransferase inhibitor that is used for the treatment of myelodysplasia 38 and possibly AML. 39,40 However, the effects of 5-aza on leukemia cell growth are not well defined.…”

Differential methylation of genes that regulate cytokine signaling in lymphoid and hematopoietic tumors

“…Aberrant promoter methylation of CpG-rich areas of promoter regions (along with the related epigenetic phenomenon of histone deacetylation) is the most frequent mechanism of tumor suppressor gene silencing in human tumors (Jones and Baylin, 2002). Three genes that regulate cytokine signaling, SHP1 (12p13-22), SOCS1 (16p12-13) and SYK (9q22), are inactivated through hypermethylation of their promoters in certain tumor types (Yoshikawa et al, 2001;Yuan et al, 2001;Oka et al, 2002). These genes are frequently methylated in lymphomas and leukemias (SHP1) (Oka et al, 2002;Chim et al, 2004), MMs (SOCS1) (Chim et al, 2004;Galm et al, 2003) and Tcell leukemias (SYK) (Goodman et al, 2003).…”

“…Methylated cell lines were cultured with Figure 1 Methylation-specific PCR (MSP) assay, RT-PCR for SHP1, SOCS1 and SYK and Western blot analyses for SHP1 and SYK in lymphoma (ly) leukemia (le) and MM cell lines. Aberrant promoter methylation of SHP1, SOCS1 and SYK was determined on bisulfite-treated DNA by the method of MSP (Herman et al, 1996), using primers for methylated sequences and methodologies previously described for SHP1 (Oka et al, 2002), SOCS1 (Galm et al, 2003) and SYK (Yuan et al, 2001). MSP assays can detect the presence of one methylated allele in the presence of 10 3 -10 4 unmethylated alleles.…”

“…Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The RT reactions were performed using SuperScript II (Invitrogen, Carlsbad, CA, USA) and Hot Star Taq DNA Polymerase (Qiagen Inc., Valencia, CA, USA) and genespecific primers (Yuan et al, 2001;Oka et al, 2002;Galm et al, 2003). Western blot analyses for protein expression were performed essentially as described previously (Shivapurkar et al, 2002b, c).…”