The Krüppel-like factor 9 (KLF9) network in HEC-1-A endometrial carcinoma cells suggests the carcinogenic potential of dys-regulated KLF9 expression

Abstract

Background: Krüppel-like factor 9 (KLF9) is a transcriptional regulator of uterine endometrial cell proliferation, adhesion and differentiation; processes essential for pregnancy success and which are subverted during tumorigenesis. The network of endometrial genes controlled by KLF9 is largely unknown. Over-expression of KLF9 in the human endometrial cancer cell line HEC-1-A alters cell morphology, proliferative indices, and differentiation, when compared to KLF9 under-expressing HEC-1-A cells. This cell line provides a unique model for identifying KLF9 downstream gene targets and signaling pathways.

Methods: HEC-1-A sub-lines differing in relative levels of KLF9 were subjected to microarray analysis to identify differentially-regulated RNAs.

Results: KLF9 under-expression induced twenty four genes. The KLF9-suppressed mRNAs encode protein participants in: aldehyde metabolism (AKR7A2, ALDH1A1); regulation of the actin cytoskeleton and cell motility (e.g., ANK3, ITGB8); cellular detoxification (SULT1A1, ABCC4); cellular signaling (e.g., ACBD3, FZD5, RAB25, CALB1); and transcriptional regulation (PAX2, STAT1). Sixty mRNAs were more abundant in KLF9 over-expressing sub-lines. The KLF9-induced mRNAs encode proteins which participate in: regulation and function of the actin cytoskeleton (COTL1, FSCN1, FXYD5, MYO10); cell adhesion, extracellular matrix and basement membrane formation (e.g., AMIGO2, COL4A1, COL4A2, LAMC2, NID2); transport (CLIC4); cellular signaling (e.g., BCAR3, MAPKAPK3); transcriptional regulation [e.g., KLF4, NR3C1 (glucocorticoid receptor), RXRalpha], growth factor/cytokine actions (SLPI, BDNF); and membrane-associated proteins and receptors (e.g., CXCR4, PTCH1). In addition, the abundance of mRNAs that encode hypothetical proteins (KLF9-inhibited: C12orf29 and C1orf186; KLF9-induced: C10orf38 and C9orf167) were altered by KLF9 expression. Human endometrial tumors of high tumor grade had decreased KLF9 mRNA abundance.

Conclusion: KLF9 influences the expression of uterine epithelial genes through mechanisms likely involving its transcriptional activator and repressor functions and which may underlie altered tumor biology with aberrant KLF9 expression.

Figure 1

Differentially expressed transcripts of HEC-1-A sub-lines. Venn diagrams summarize the number of differentially expressed genes noted between 2AS and 4S sub-lines, between 3AS and 9S sub-lines, and those in common for both comparisons (final annotated gene lists are presented in additional file 1: Table 1 and additional file 2: Table 2). There were greater numbers of genes induced than repressed in concert with relative KLF9 expression.

Figure 2

Hierarchical clustering of differentially expressed RNAs. The microarray data for mRNAs that were identified to be differentially expressed between S and AS sub-lines (additional file 1: Table 1 and additional file 2: Table 2) were subjected to hierarchical clustering. The transcript profiles were very similar for both S sub-lines (each run in duplicate); whereas the two AS sub-lines differed from each other and from the S sub-lines. Lower case letters signify duplicate microarrays for each sub-line.

Figure 3

Quantitative RT-PCR of KLF9 mRNA in human endometrium and endometrial tumors. A normalized cDNA panel of human endometrial tumors was obtained from OriGene Technologies, Inc. This panel was comprised of cDNAs from n = 6 of normal (N) endometria, n = 9 of Stage I tumors, n = 8 of Stage II tumors, n = 19 of Stage III tumors, and n = 6 of Stage IV tumors. Shown are box plots (median, upper and lower quartiles, minimum and maximum data values) of relative abundance of KLF9 mRNA for tumors (delineated by stage and tumor type: endometrioid, serous). Sample numbers are indicated. ANOVA indicated no differences in mRNA abundance between any of the individual stages. However, a significant difference between combined stages (normal plus stage I vs. stages II, III, and IV) was noted by the Mann-Whitney Rank Sum test.