Abstract
Germline and somatic promoter hypermethylation of KLLN has been found in diverse heritable and sporadic cancers, respectively. KLLN has many identified tumor suppressor functions, and when first reported, was thought to be exclusively nuclear. Here, we report on KLLN localization in both the nucleus and cytoplasm and the identification of a putative nuclear export signal (NES) sequence. KLLN overexpression in colon and breast cancer cells showed both nuclear and cytoplasmic presence. Inhibition of the CRM1 export pathway increased nuclear sequestration of KLLN, confirming the prediction of an NES sequence. Point mutations introduced in the predicted NES sequence decreased the strength of the NES and increased the nuclear sequestration of KLLN. Contrary to expectations, the transcription regulation and cellular proliferation functions of KLLN were unaffected by increased KLLN nuclear sequestration. Instead, increased nuclear KLLN correlated with increased nuclear sequestration of TRIM25 and decreased inhibitory phosphorylation of MDM2. Computational analysis of The Cancer Genome Atlas (TCGA) dataset showed positive correlation among KLLN, TRIM25 and MDM2 expression; pathway analysis of the common genes downstream of these three genes revealed protein degradation as one of the top canonical pathways. Together, our observations suggest that CRM1 pathway-based nuclear export of KLLN may impact proteasomal degradation.
Highlights
KLLN is a tumor suppressor protein discovered in 2008 when researchers were searching for potential new targets of p53 that were involved in S-phase cell cycle checkpoint regulation
We show that KLLN possesses a nuclear export signal (NES) that helps it exit the nucleus and that the likely role KLLN plays in trafficking proteins between the nuclear and cytoplasmic compartments may relate to proteasomal degradation
To empirically confirm the computational data, we used plasmid-based transfection of GFP and FLAGtagged KLLN plasmids followed by immunoblotting to show that KLLN localizes to both the nucleus and cytoplasm in breast and colon cancer cell lines (Figure 1A and 1B)
Summary
KLLN is a tumor suppressor protein discovered in 2008 when researchers were searching for potential new targets of p53 that were involved in S-phase cell cycle checkpoint regulation. In PTEN mutation negative CS/CSL, KLLN germline promoter hypermethylation is observed in up to 35% of patients and is associated with three-fold increased prevalence of breast carcinomas and two-fold increased prevalence of renal cell carcinomas [2, 5, 6]. Recent studies looking into somatic promoter hypermethylation in ductal carcinoma in situ of the male breast have reported the presence of KLLN in a cluster of genes that are frequently methylated [7]. In oral squamous cell carcinomas, somatic KLLN promoter methylation was www.oncotarget.com found to be associated with relapse or development of metastasis in clinical follow-up [8]. KLLN could broadly contribute to both cancer susceptibility and sporadic carcinogenesis
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