Development of LHX6-inducible system in PK-1 and PK-9 cells

Epigenetic gene silencing by aberrant DNA methylation leads to loss of key cellular pathways in tumorigenesis. DNA methylation-mediated silenced genes in pancreatic cancer were searched by methyl-CpG targeted transcriptional activation (MeTA) method and LHX6 (LIM homeobox 6), a transcription factor involved in embryogenesis and head development, was selected as one of candidate genes. LHX6 was downregulated in most pancreatic cancer cell lines (83%: 10/12) mainly through promoter hypermethylation and histone deacetylation. Furthermore, LHX6 was also methylated in primary pancreatic cancers in a tumor-specific manner (57%: 16/28). In order to assess the biological significance of LHX6 in pancreatic tumorigenesis, we first performed colony formation assay and found that LHX6 re-expression inhibited colony formation in pancreatic cancer cell lines. Similarly, inducible expression of LHX6 inhibited cell proliferation and migration in LHX6 low-expressing pancreatic cancer cell lines. On the other hand, knockdown of LHX6 accelerated cell proliferation in LHX6 high-expressing pancreatic cancer cell lines. Our present results suggest that epigenetic inactivation of LHX6 plays an important role in pancreatic tumorigenesis by promoting cell proliferation. Two pancreatic cancer-derived cell lines, PK-1_LHX6#13 and PK-9_LHX6#4, that allow tetracycline-regulated LHX6 expression were constructed. The RNAs from PK-1_LHX6#13 and PK-9_LHX6#4 with (4 days treatment) or without tetracycline were prepared and analyzed by using Agilent whole human genome microarray (4×44K) v2.