Interaction between the bone morphogenetic proteins and Ras/MAP-kinase signalling pathways in lung cancer

Abstract

Bone morphogenetic proteins (BMPs) are an integral component of the TGFbeta superfamily, responsible for regulation of cell proliferation, differentiation, migration and programmed cell death in a variety of cell types. The BMPs transduce their signals directly through the SMAD family of proteins but they also have been reported to interact with the MAPK and Erk pathways. Inactivation of the BMP pathway genes has been implicated as important in several cancers. Recent work has shown that BMP3b is epigenetically inactivated in cancer and suggests that BMP6 can be epigenetically inactivated. We investigated whether BMP6 is epigenetically inactivated in cell lines and whether BMP3b and BMP6 are epigenetically inactivated in non-small-cell lung cancer (NSCLC). We also studied the relationship between BMP methylation and k-ras mutation. Here, we demonstrate that the BMP3b and BMP6 genes are common targets of epigenetic inactivation in NSCLC, and that they are significantly more likely to be concurrently inactivated (P=0.009). Furthermore, this coinactivation of BMP3b and BMP6 is significantly associated with mutation of k-ras codon 12 in lung cancer (P=0.003); those with a k-ras mutation were six times more likely to have concurrent methylation of these BMP loci. Hence, these data suggest that concurrent inactivation of the BMP and activation of the Ras signalling pathways are important in lung carcinogenesis.

Figure 1

BMP6 is epigenetically inactivated in cancer cell lines. (A) BMP6 cDNA was amplified using an annealing temperature of 60°C over 35 cycles with the following primers: 5′-ACA GCA TAA CAT GGG GCT TC-3′ (sense) and 5′-CTC GGG GTT CAT AAG GTG AA-3′ (antisense). The WTK1 and HT cell lines did not express BMP6. (B) WTK1 cells were treated with 1 μl 5-aza-deoxycytadine in PBS or PBS alone for 3 days and then harvested. BMP6 is re-expressed in the WTK1 cells. (C) The BMP6 gene promoter methylation status was determined by amplifying bisulphite-modified DNA using primers specific to methylated (M) and unmethylated (U) DNA. The DNA was amplified using an annealing temperature of 60°C over 38 cycles with the following primers: (M) 5′-GGT TTG TTG GGT AGT CGG G-3′ (sense) and GCC CCT CCC CAA ATC G-3′ (antisense) and (U) 5′-TTG GGT AGT TGG GTG ATT GTT-3′ (sense) and 5′-ACA CCC CTC CCC AAA TCA-3′ (antisense).