CKS1B, overexpressed in aggressive disease, regulates multiple myeloma growth and survival through SKP2- and p27Kip1-dependent and -independent mechanisms

Abstract

Overexpression of CKS1B, a gene mapping within a minimally amplified region between 153 to 154 Mb of chromosome 1q21, is linked to a poor prognosis in multiple myeloma (MM). CKS1B binds to and activates cyclin-dependent kinases and also interacts with SKP2 to promote the ubiquitination and proteasomal degradation of p27(Kip1). Overexpression of CKS1B or SKP2 contributes to increased p27(Kip1) turnover, cell proliferation, and a poor prognosis in many tumor types. Using 4 MM cell lines harboring MAF-, FGFR3/MMSET-, or CCND1-activating translocations, we show that lentiviral delivery of shRNA directed against CKS1B resulted in ablation of CKS1B mRNA and protein with concomitant stabilization of p27(Kip1), cell cycle arrest, and apoptosis. Although shRNA-mediated knockdown of SKP2 and forced expression of a nondegradable form of p27(Kip1) (p27(T187A)) led to cell cycle arrest, apoptosis was modest. Of importance, while knockdown of SKP2 or overexpression of p27(T187A) induced cell cycle arrest in KMS28PE, an MM cell line with biallelic deletion of CDKN1B/p27(Kip1), CKS1B ablation induced strong apoptosis. These data suggest that CKS1B influences myeloma cell growth and survival through SKP2- and p27(Kip1)-dependent and -independent mechanisms and that therapeutic strategies aimed at abolishing CKS1B function may hold promise for the treatment of high-risk disease for which effective therapies are currently lacking.

Figure 1

CKS1B mRNA and protein expression are inversely correlated with p27^Kip1 protein levels in primary disease. (A) CKS1B and (B) CDKN1B (p27^Kip1) gene expression signal in 1000-unit increments is plotted on the y-axis. Primary myeloma with CKS1B expression in quartile 1 (n = 13) and quartile 4 (n = 14) and MM cell lines (n = 7) are grouped and plotted from left to right along the x-axis. Each bar represents a sample, and the height indicates the level of gene expression in each sample. (C) CKS1B, (D) p27^Kip1, and (E) Histone 1 levels were evaluated by Western blot analysis of nuclear fractions derived from the same aliquots of plasma cells used in panels A-B. Samples are ordered from left to right in the exact same order in all panels.

Figure 2

Effects of silencing CKS1B or SKP2 and overexpressing a nondegradable form of p27^Kip1 on MM cell growth and survival. CKS1B, SKP2, and CDKN1B (p27^Kip1) mRNA expression was evaluated by microarray analysis 5 days after lentiviral infection in (A) JJN3, (B) OCI-MY5, and (C) XG-1 cells expressing a nonspecific scrambled shRNA (SCRAM), CKS1B shRNA, or SKP2 shRNA. All experiments were performed in duplicate, and the results were expressed as the mean ± standard error. Western blot analysis on nuclear fractions of the same aliquots of cells shown in panels A, B, or C confirmed that CKS1B shRNA and SKP2 shRNA efficiently inhibited CKS1B and SKP2 protein expression, respectively, and increased p27^T187A levels in (D) JJN3, (E) OCI-MY5, and (F) XG-1. Cullin 1A (Cul1A) expression was used as control for the specificity of CKS1B and SKP2 action. p27^T187A overexpression was confirmed in all 3 cell lines 5 days after lentiviral infection, compared to the control cells infected with an empty vector (EV). Histone 1 was used as a loading control. Effects of CKS1B or SKP2 shRNA and p27^T187A overexpression on (G,I,K) cell proliferation and (H,J,L) cell viability in (G-H) JJN3, (I-J) OCI-MY5, and (K-L) XG-1. Total number of cells and cell viability were evaluated every day after lentiviral infection by trypan blue exclusion. Error bars represent standard error of the mean for 2 independent experiments.

Figure 3

Silencing of CKS1B induces apoptosis by activating caspase-3 and PARP. (A) Cell cycle distribution and apoptosis were evaluated by flow cytometry analysis performed 5 days after lentiviral infection in JJN3 cells expressing a scrambled sequence (SCRAM), CKS1B shRNA, SKP2 shRNA, or p27^T187A cDNA. Note that silencing of CKS1B induced cell cycle arrest in G₀-G₁ phase and dramatic increase in the percentage of cells with sub-G₀ DNA content (indicative of apoptosis). SKP2 shRNA deregulated the G₁-S transition more strongly than CKS1B shRNA, but caused a modest increase of apoptotic cells; the overexpression of p27^T187A had no affect on apoptosis, but resulted in a significantly higher percentage of cells in G₂-M phase. The same analysis shown in panel A was performed in (B) OCI-MY5 and (C) XG-1. (D) Caspase-3 (active form, p17) and PARP activation was evaluated by Western blot analysis performed in the same aliquots of cells used in panels A-C. The following conditions were analyzed: (1) positive control (myeloma cells treated with bortezomib at 10 nM for 24 hours); (2) positive control + Z-VAD-fmk; (3) negative control (myeloma cells without any treatment); (4) CKS1B shRNA; (5) CKS1B shRNA + Z-VAD-fmk; (6) SCRAM; (7) SCRAM + Z-VAD-fmk; (8) empty vector; (9) p27^T187A cDNA; (10) SKP2 shRNA. Note that the activation of caspase-3 and PARP in cells expressing CKS1B shRNA could be abrogated by pretreatment with the pan-caspase inhibitor Z-VAD-fmk, indicating a caspase-dependent mechanism of apoptosis.

Figure 4

Silencing of CKS1B induces apoptosis independent of p27^Kip1 regulation in KMS28PE cell line. (A) CKS1B, SKP2, and CDKN1B (p27^Kip1) mRNA expression was evaluated by microarray analysis 5 days after lentiviral infection in KMS28PE cells expressing a nonspecific scrambled shRNA (SCRAM), CKS1B shRNA, or SKP2 shRNA. Note that CDKN1B expression is absent in KMS28PE, compared to the mean expression value of the other cell lines shown in Figure 2A-C. All experiments were performed in duplicate and the results were expressed as the mean ± standard error. (B) Western blot analysis on nuclear fractions of the same aliquots of cells shown in panel A confirmed that CKS1B shRNA and SKP2 shRNA efficiently inhibited CKS1B and SKP2 protein expression, respectively, and did not increase p27^Kip1 levels that remained absent. Cullin 1A (Cul1A) expression was used as control for the specificity of CKS1B and SKP2 action. p27^T187A overexpression was confirmed in KMS28PE 5 days after lentiviral infection, compared to the control cells infected with an empty vector (EV). Histone 1 was used as a loading control. Effects of CKS1B or SKP2 shRNA and p27^T187A overexpression on (C) cell proliferation and (D) cell viability in KMS28PE. (E) Cell cycle distribution and apoptosis were evaluated by flow cytometry analysis performed 5 days after lentiviral infection in KMS28PE cells expressing a scrambled sequence (SCRAM), CKS1B shRNA, SKP2 shRNA, or p27^T187A cDNA. Note that silencing of CKS1B induced a dramatic increase in the percentage of cells with sub-G₀ DNA content (indicative of apoptosis); SKP2 shRNA caused a modest increase of apoptotic cells, and overexpression of p27^T187A had no affect on apoptosis but resulted in a significantly higher percentage of cells in G₂-M phase. (F) Caspase-3 (active form, p17) and PARP activation was evaluated by Western blot analysis performed in the same aliquots of cells used in panel E. The following conditions were analyzed: (1) positive control (myeloma cells treated with bortezomib at 10 nM for 24 hours); (2) positive control + Z-VAD-fmk; (3) negative control (myeloma cells without any treatment); (4) CKS1B shRNA; (5) CKS1B shRNA + Z-VAD-fmk; (6) SCRAM; (7) SCRAM + Z-VAD-fmk; (8) empty vector; (9) p27^T187A cDNA; (10) SKP2 shRNA. Note that the activation of caspase-3 and PARP in cells expressing CKS1B shRNA could be abrogated by the pretreatment with the pan-caspase inhibitor Z-VAD-fmk, indicating a caspase-dependent mechanism of apoptosis.