Cell adhesion induces p27Kip1-associated cell-cycle arrest through down-regulation of the SCFSkp2 ubiquitin ligase pathway in mantle-cell and other non-Hodgkin B-cell lymphomas

Abstract

Mounting evidence suggests that dynamic interactions between a tumor and its microenvironment play a critical role in tumor development, cell-cycle progression, and response to therapy. In this study, we used mantle cell lymphoma (MCL) as a model to characterize the mechanisms by which stroma regulate cell-cycle progression. We demonstrated that adhesion of MCL and other non-Hodgkin lymphoma (NHL) cells to bone marrow stromal cells resulted in a reversible G(1) arrest associated with elevated p27(Kip1) and p21 (WAF1) proteins. The adhesion-mediated p27(Kip1) and p21 increases were posttranslationally regulated via the down-regulation of Skp2, a subunit of SCF(Skp2) ubiquitin ligase. Overexpression of Skp2 in MCL decreased p27(Kip1), whereas inhibition of Skp2 by siRNA increased p27(Kip1) and p21 levels. Furthermore, we found cell adhesion up-regulated Cdh1 (an activating subunit of anaphase-promoting complex [APC] ubiquitin ligase), and reduction of Cdh1 by siRNA induced Skp2 accumulation and hence p27(Kip1) degradation, thus implicating Cdh1 as an upstream effector of the Skp2/p27(Kip1) signaling pathway. Overall, this report, for the first time, demonstrates that cell-cell contact controls the tumor cell cycle via ubiquitin-proteasome proteolytic pathways in MCL and other NHLs. The understanding of this novel molecular pathway may prove valuable in designing new therapeutic approaches for modifying tumor cell growth and response to therapy.

Figure 1

Cell adhesion to bone marrow stroma induces a reversible cell-cycle arrest and increases p27^Kip protein levels. Jeko-1 (A) or SUDH-10 (B) lymphoma cells (10⁵/mL) were incubated for 12 hours either in the absence of HS-5 bone marrow stromal cells (Sus), or on a confluent HS-5 monolayer (HS5-adhesion), or in the same well with a confluent HS-5 monolayer but separated by cell culture inserts (HS-5 + Transwell). (C) Jeko-1 cells were resuspended for 12 hours after adhering to HS-5 (Resuspension). After the indicated time, lymphoma cells were followed by pulsing with BrdU for 40 minutes and staining with anti-BrdU-FITC for detection of S-phase cells and PI for cell-cycle distribution. Data shown are the mean of 3 experiments. (D) Direct cell-cell contact but not soluble factor(s) increased p27^Kip1 expression analyzed by Western blot in Jeko-1 and SUDH-10 lymphoma cells. All data are representative of at least 4 experiments (means ± SD).

Figure 2

Cell adhesion–mediated p27^Kip1 protein levels are posttranslationally regulated through down-regulation of the SCF^Skp2 ubiquitin ligase Skp2. (A) An RPA for gene expression of p27^Kip1, p21, and other cell-cycle–related molecules in Jeko-1 and SUDH-10 cells adhered to HS-5 versus those placed in suspension media. (B) The p27^Kip1 and Skp2 expression in Jeko-1 cells in suspension (Sus) versus HS-5 adhesion (HS5-Ad) analyzed by Western blot. Cell-cycle distribution (C) and p27^Kip1 and Skp2 expression (D) 24 hours after transfection with either Skp2 siRNA (siSkp2) or control siRNA (siCont) in Jeko-1 cells in suspension. (E) Cyclin D1 expression in Jeko-1 and SUDH-10 lymphoma cells in suspension (Sus) versus HS-5 adhesion (HS5-Ad) analyzed by Western blot. All data are representative of at least 3 experiments (means ± SD).

Figure 3

Cell adhesion–mediated down-regulation of Skp2 requires Cdh1. (A) The Cdh1 expression in Jeko-1 cells in suspension (Sus) versus HS-5 adhesion (HS5-Ad) analyzed by Western blot. Cell-cycle distribution (B) and protein levels of p27^Kip1, Skp2, and Cdh1 (C) 24 hours after transfection with Skp2 siRNA (siSkp2), Cdh1 siRNA (siCdh1), or control siRNA (siCont) in Jeko-1 lymphoma cells in suspension. All data are representative of at least 3 experiments (means ± SD).

Figure 4

p27^Kip1 depletion, Skp2 overexpression, and Cdh1 knock down abolished cell-adhesion–mediated p27^Kip1 induction and cell-cycle arrest. (A) The p27^Kip1 expression in control siRNA entering RISC-transfected (siCont.RISC[ + ]) or siP27^Kip1 RNA-transfected (siP27) Jeko-1 cells in suspension (Sus) versus 12 hours of HS-5 adhesion (HS5-Ad) analyzed by Western blot, and (B) corresponding cell-cycle distribution in suspension (Sus) versus HS-5 adhesion (HS5-Ad) analyzed by flow cytometry. (C) The Skp2 and p27^Kip1 expression in nontransfected [pCont(−)] Jeko-1 cells in suspension (Sus) versus HS-5 adhesion (HS5-Ad) or stably mock (pCont( + ))- or plasmid Skp2-transfected (pSkp2) Jeko-1 cells in suspension versus to HS-5 adhesion for 12 hours analyzed by Western blot and (D) corresponding cell-cycle distribution in stably mock- or plasmid Skp2-transfected Jeko-1 cells in suspension (pCont( + )-Sus or pSkp2-Sus) versus HS-5 adhesion (pCont( + )-Ad or pSkp2-Ad) for 12 hours analyzed by flow cytometry. (E) Cdh1, Skp2, and p27^Kip1 expressions in nonsilencing siControl RNA-transfected (siCont) or siCdh1 RNA-transfected (siCdh1)-Jeko-1 cells in suspension (Sus) versus HS-5 adhesion (HS5-Ad) for 12 hours analyzed by Western blot, and (F) corresponding cell-cycle distribution in control siRNA- or Cdh1 siRNA-transfected Jeko-1 cells in suspension (siCont-Sus or siCdh1-Sus) versus HS-5 adhesion (siCont-Ad or siCdh1-Ad) for 12 hours analyzed by flow cytometry. Each blot is representative of 3 experiments, and the bar graphs show means plus or minus SD of 3 experiments.

Figure 5

The molecular mechanisms for cell-adhesion–mediated Skp2 and Cdh1 changes. (A,B) Effect of cell adhesion on Skp2 degradation. Jeko-1 cells were treated with cycloheximide (100 μM) with and without HS-5 cells adhesion for the indicated time periods. At the times indicated, cells were lysed, and Skp2 protein levels were determined by Western blot analysis (A) and quantified (from 3 independent experiments) using densitometry. The data shown (B) represent mean values and standard deviations of the average percentage of Skp2, compared with the amount of Skp2 at time 0 for each treatment group. (C,D) Cell-adhesion–induced changes of Skp2 and CDH1 mRNA expressions measured by real-time qRT-PCR. Fold values were obtained by externally standardizing against identical amplifications in Jeko-1 and SUDH-10 cells in suspension versus HS-5 adhesion and by internally standardizing against GAPDH in each cell line. The mean values and standard deviations from 3 independent experiments are shown. Student t test was used for statistical analysis. *P < .05.

Figure 6

Cell-adhesion–induced p27^Kip1-associated cell-cycle arrest through down-regulating the SCF^Skp2 ubiquitin ligase pathway in primary MCL and other non-Hodgkin B-cell lymphomas. (A,B) The Cdh1, Skp2, and p27^Kip1 expressions in primary MCL (A) and DLBCL (B) cells in suspension (Sus) versus HS-5 adhesion (HS5-Ad) were analyzed by Western blot. (C) Cell-cycle distribution in DLBCL (DLBL0401) cells in suspension (Sus), or on a confluent HS-5 monolayer (HS5-Ad; 12 hours).

Figure 7

Model for cell-adhesion–mediated cell-cycle arrest. Hypothetically ordered series of signaling events: cell-adhesion–dependent activation of APC/Cdh1 ubiquitin ligase complex, increased ubiquitination and degradation of Skp2, subsequent decreased ubiquitination and degradation of p27^Kip1, and ultimately p27^Kip1 accumulation, resulting in cell-cycle arrest.