Caveolin-1-mediated expression and secretion of kallikrein 6 in colon cancer cells - PubMed (original) (raw)

Caveolin-1-mediated expression and secretion of kallikrein 6 in colon cancer cells

Rebecca S Henkhaus et al. Neoplasia. 2008 Feb.

Abstract

Kallikreins are secreted proteases that may play a functional role and/or serve as a serum biomarker for the presence or progression of certain types of cancers. Kallikrein 6 (KLK6) has been shown to be upregulated in several types of cancers, including colon. The aims of this study were to elucidate pathways that influence KLK6 gene expression and KLK6 protein secretion in the HCT116 human colon cancer cells. Our data indicate a central role for caveolin-1 (CAV-1), the main structural protein of caveolae, in both KLK6 gene expression and protein secretion. Sucrose gradient subcellular fractionation reveals that CAV-1 and KLK6 colocalize to lipid raft domains in the plasma membrane of HCT116 cells. Furthermore, we show that CAV-1, although it does not directly interact with the KLK6 molecule, enhances KLK6 secretion from the cells. Deactivation of CAV-1, through SRC-mediated phosphorylation, decreased KLK6 secretion. We also demonstrate that, in colon cancer cells, CAV-1 increased the amount of phosphorylated AKT in cells by inhibiting the activity of the AKT-negative regulators PP1 and PP2A. This study demonstrates that proteins such as CAV-1 and AKT, which are known to be altered in colon cancer, affect KLK6 expression and KLK6 secretion.

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Figures

Figure 1

Downregulation of CAV-1 in HCT116 cells decreases the levels of KLK6 mRNA and KLK6 secretion. (A) Whole-cell lysates were collected from HCT116 CAV-1 mock and CAV-1 AS cells after 48 hours of growth under normal conditions (regular media with 10% FBS). Western blot analysis for CAV-1 was performed and β-actin was used as a loading control. (B) Real-time reverse transcription-PCR analysis was performed on RNA isolated from cells after 48 hours of growth under normal conditions (*P ≤ .05). (C) ELISA analysis for secreted KLK6 in media collected from HCT116 CAV-1 mock and AS cells 24, 48, and 72 hours after plating in normal growth conditions (*P ≤ .02).

Figure 2

Sucrose gradient cell fractionation reveals that KLK6 associates with lipid raft domains in CAV-1 expressing cells. (A and B) Equal amounts from each of the 12 sucrose gradient fractions obtained from HCT116 CAV-1 Mock cells (A) and HCT116 CAV-1 AS cells (B) were loaded and run on a 12.5% SDS-PAGE gel, and immunoblots for KLK6, CAV-1, and Flot-1 were performed. (C) Protein localization on sucrose gradient in HCT116 CAV-1 Mock cells. (D) Protein localization on sucrose gradient in HCT116 CAV-1 AS cells. (E) KLK6 is associated with the membrane fraction but does not interact with CAV-1. CAV-1 was immunoprecipitated from the cytosolic (C) and membrane (M) fractions isolated from HCT116 CAV-1 Mock and CAV-1 AS cells as described in Materials and Methods. Western blot analysis for KLK6, CAV-1, and β-actin (used as a loading control) was performed in immunoprecipitates (IP:CAV-1) and in unprecipitated whole fractions (WF). For immunoprecipitates, 500 µg of protein was used; for whole fractions, each lane was loaded with 80 µg of protein.

Figure 3

HCT116 cells with constitutively active SRC have elevated levels of phosphorylated CAV-1, leading to less KLK6 secretion. (A) Western blot analysis was performed on whole-cell lysates from HCT116 SRC-mock and HCT116 SRC531 cells collected 48 hours after plating in normal growth conditions. (B) ELISA analysis for secreted KLK6 was performed on conditioned media collected from HCT116 SRC-mock and HCT116 SRC531 cells 48 hours after plating in normal growth conditions (*P ≤ .001).

Figure 4

Increased KLK6 expression correlates to levels of phospho-AKT and the phosphatases PP1 and PP2A. (A) Western blot analyses of phospho-AKT and total AKT were performed on whole-cell lysates collected 48 hours after plating in normal growth conditions. (B) Western blot analyses for PP1 and PP2A were performed on whole-cell lysates collected from cells 48 hours after plating in normal growth conditions. β-Actin was used as a loading control.

Figure 5

Inhibition of AKT leads to decreased KLK6 mRNA and secreted KLK6 protein. (A) Real-time PCR analysis for the detection of KLK6 mRNA was performed using RNA isolated from cells treated with either the AKT inhibitor LY294002 or the vehicle control DMSO (*P ≤ .03). The exact treatment protocol is outlined in the Materials and Methods section. (B) ELISA analysis was performed on conditioned media collected after 48 hours of treatment with LY294002 (*P ≤ .01).

Figure 6

Proposed model of KLK6 expression and secretions: This proposed model unites the pathways regulating KLK6 gene expression and KLK6 secretion. We demonstrate that AKT plays the most pivotal role in KLK6 gene expression. AKT activity is negatively regulated by phosphatases PP1 and PP2A, which are in turn controlled at least in part by CAV-1. Colon cancer cells, which express CAV-1, express and secrete significantly more KLK6 than isogenic cells lines lacking CAV-1 expression. SRC kinase phosphorylates CAV-1, and this phosphorylation causes the decrease in KLK6 secretion.

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Caveolin-1-mediated expression and secretion of kallikrein 6 in colon cancer cells - PubMed (original) (raw)