Emerging roles of Kruppel-like factor 6 and Kruppel-like factor 6 splice variant 1 in ovarian cancer progression and treatment

Abstract

Epithelial ovarian cancer is one of the most lethal gynecologic cancers and the fifth most frequent cause of female cancer deaths in the United States. Despite dramatic treatment successes in other cancers through the use of molecular agents targeted against genetically defined events driving cancer development and progression, very few insights into epithelial ovarian cancer have been translated from the laboratory to the clinic. If advances are to be made in the early diagnosis, prevention, and treatment of this disease, it will be critical to characterize the common and private (personalized) genetic defects underlying the development and spread of epithelial ovarian cancer. The tumor suppressor Kruppel-like factor 6 and its alternatively spliced, oncogenic isoform, Kruppel-like factor 6 splice variant 1, are members of the Kruppel-like zinc finger transcription factor family of proteins, which have diverse roles in cellular differentiation, development, proliferation, growth-related signal transduction, and apoptosis. Inactivation of Kruppel-like factor 6 and overexpression of Kruppel-like factor 6 splice variant 1 have been associated with the progression of a number of human cancers and even with patient survival. This article summarizes our recent findings demonstrating that a majority of epithelial ovarian cancer tumors have Kruppel-like factor 6 allelic loss and decreased expression coupled with increased expression of Kruppel-like factor 6 splice variant 1. The targeted reduction of Kruppel-like factor 6 in ovarian cancer cell lines results in marked increases in cell proliferation, invasion, tumor growth, angiogenesis, and intraperitoneal dissemination in vivo. In contrast, the inhibition of Kruppel-like factor 6 splice variant 1 decreases cellular proliferation, invasion, angiogenesis, and tumorigenicity; this provides the rationale for its potential therapeutic application. These results and our recent demonstration that the inhibition of Kruppel-like factor 6 splice variant 1 can dramatically prolong survival in a preclinical mouse model of ovarian cancer are reviewed and discussed.

Fig 1

Loss of KLF6 expression in epithelial ovarian cancer tumors with LOH. Abbreviations: KLF6, Kruppel-like factor 6; LOH, loss of heterozygosity; mRNA, messenger RNA.

Fig 2

KLF6 expression in OSE brushings versus select housekeeping genes. (A) KLF6 microarray signal intensity values were averaged for 10 OSE brushings and compared to the average expression levels of 5 housekeeping genes: GUSB, ACTA2, ACTB, PPIH, and PPIA. The KLF6 expression level was comparable to the housekeeping gene levels, and this indicates that KLF6 is reliably expressed in ovarian epithelial cells. (B) KLF6 expression in 76 stage III EOC samples versus KLF6 expression in OSE brushings. The KLF6 expression in each microdissected EOC sample was compared to the average expression in all 10 OSE samples. The fold changes were normalized to zero. Abbreviations: ACTA2, actin alpha 2 smooth muscle aorta; ACTB, actin beta; EOC, epithelial ovarian cancer; GUSB, glucuronidase beta; HOSE, human ovarian surface epithelial; KLF6, Kruppel-like factor 6; OSE, ovarian surface epithelium; PPIA, peptidylprolyl isomerase A (cyclophilin A); PPIH, peptidylprolyl isomerase H (cyclophilin H).

Fig 3

KLF6-SV1 is overexpressed in nearly all epithelial ovarian cancer, regardless of the LOH status, and its expression increases with a higher tumor grade. Abbreviations: KLF6-SV1, Kruppel-like factor 6 splice variant 1; LOH, loss of heterozygosity.

Fig 4

Invasion capacity in small interfering RNA stable cells. The inhibition of KLF6-SV1 decreases tumor invasion and metastasis. The upper panels are representative photomicrographs of the underside of a Matrigel insert stained with 4′,6-diamidino-2-phenylindole. The lower panel shows the number of cells per visual field (400× magnification). The cell number was determined from an analysis of 4 representative fields and 3 independent experiments. The statistical analysis was performed with the Student t test with a 2-tailed distribution and 2 samples of equal variance. Abbreviations: si-KLF6, pSUPER small interfering Kruppel-like factor 6; si-luc, pSUPER small interfering luciferase; si-SV1, pSUPER small interfering splice variant 1.

Fig 5

The inhibition of Kruppel-like factor 6 increases intraperitoneal tumor dissemination and vascular endothelial growth factor expression. Abbreviations: SD, standard deviation; si-KLF6, pSUPER small interfering Kruppel-like factor 6; si-luc, pSUPER small interfering luciferase.

Fig 6

The inhibition of Kruppel-like factor 6 and Kruppel-like factor 6 splice variant 1 has markedly antagonistic effects on E-cadherin expression. Abbreviations: mRNA, messenger RNA; si-KLF6, pSUPER small interfering Kruppel-like factor 6; si-luc, pSUPER small interfering luciferase; si-SV1, pSUPER small interfering splice variant 1.