The in vitro and in vivo effects of re-expressing methylated von Hippel-Lindau tumor suppressor gene in clear cell renal carcinoma with 5-aza-2'-deoxycytidine.
OBJECTIVE
Clear cell renal carcinoma (ccRCC) is strongly associated with loss of the von Hippel-Lindau (VHL) tumor suppressor gene. The VHL gene is functionally lost through hypermethylation in up to 19% of sporadic ccRCC cases. We theorized that re-expressing VHL silenced by methylation in ccRCC cells, using a hypo-methylating agent, may be an approach to treatment in patients with this type of cancer. We test the ability of two hypo-methylating agents to re-express VHL in cell culture and in mice bearing human ccRCC and evaluate the effects of re-expressed VHL in these models.


METHODS
Real-time reverse transcription-PCR was used to evaluate the ability of zebularine and 5-aza-2'-deoxycytidine (5-aza-dCyd) to re-express VHL in four ccRCC cell lines with documented VHL gene silencing through hypermethylation. We evaluated if the VHL re-expressed after hypo-methylating agent treatment could recreate similar phenotypic changes in ccRCC cells observed when the VHL gene is re-expressed via transfection in cell culture and in a xenograft mouse model. Finally we evaluate global gene expression changes occurring in our cells, using microarray analysis.


RESULTS
5-Aza-dCyd was able to re-express VHL in our cell lines both in culture and in xenografted murine tumors. Well described phenotypic changes of VHL expression including decreased invasiveness into Matrigel, and decreased vascular endothelial growth factor and glucose transporter-1 expression were observed in the treated lines. VHL methylated ccRCC xenografted tumors were significantly reduced in size in mice treated with 5-aza-dCyd. Mice bearing nonmethylated but VHL-mutated tumors showed no tumor shrinkage with 5-aza-dCyd treatment.


CONCLUSIONS
Hypo-methylating agents may be useful in the treatment of patients having ccRCC tumors consisting of cells with methylated VHL.
Alleman
Wade G
WG
Howard Hughes Medical Institute, Chevy Chase, MD, USA.
Tabios
Ray L
RL
Chandramouli
Gadisetti V R
GV
Aprelikova
Olga N
ON
Torres-Cabala
Carlos
C
Mendoza
Arnulfo
A
Rogers
Craig
C
Rodgers
Craig
C
Sopko
Nikolai A
NA
Linehan
W Marston
WM
Vasselli
James R
JR
D018262:Adenocarcinoma, Clear Cell / Q000235:genetics* / Q000473:pathology; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D003562:Cytidine / Q000031:analogs & derivatives; D019175:DNA Methylation; D000077209:Decitabine; D004195:Disease Models, Animal; D020868:Gene Silencing; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics* / Q000473:pathology; D051379:Mice; D011741:Pyrimidine Nucleosides / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D051794:Von Hippel-Lindau Tumor Suppressor Protein
D000964:Antimetabolites, Antineoplastic; D011741:Pyrimidine Nucleosides; D025521:Tumor Suppressor Proteins; D003562:Cytidine; D000077209:Decitabine; C009131:pyrimidin-2-one beta-ribofuranoside; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human; D001374:Azacitidine