Vitamin D arrests thyroid carcinoma cell growth and induces p27 dephosphorylation and accumulation through PTEN/akt-dependent and -independent pathways

Abstract

We investigated the effects of 1,25-dihydroxycholecalciferol vitamin D(3) (VD) and its noncalciomimetic analog EB1089 on thyroid carcinoma cell growth. VD and EB1089 exhibited anti-proliferative effects in a dose-dependent manner as determined by [(3)H]thymidine incorporation and MIB-1 immunolabeling. VD or EB1089 resulted in similar G(1)-phase arrest. Neither apoptosis nor differentiation was affected. VD and EB1089 induced increased nuclear protein expression of the cyclin-dependent kinase inhibitor, p27(kip1) (p27). VD/EB1089 effects paralleled but were not additive to those of the proteasome inhibitor LLnL, consistent with reduced p27 degradation. As p27 phosphorylation and association with Skp2 is a key step in its degradation, we examined the effects of VD/EB1089 on this reaction. Despite increased total p27, the pThr content of p27 remained unaffected, an effect confirmed by diminished association with Skp2 as well as in situ phosphorylation. Moreover, phosphatase inhibition abrogated the effect of VD/EB1089 on p27 accumulation consistent with a role for phosphatase action in mediating this VD effect. Although VD/EB1089 resulted in comparable increases in p27 in WRO and NPA cells, only WRO but not NPA cells demonstrated a change in the phosphatase PTEN and its downstream target pAkt/PKB in response to VD/EB1089. Transfection of PTEN resulted in p27 accumulation and was partially additive to the effect of VD/EB1089. Moreover, treatment with PI-3 kinase inhibitors decreased pAkt/PKB and increased p27 in both WRO and NPA cells highlighting the potential role of this downstream pathway in regulating p27 in the thyroid. These findings point to a novel mechanism of action for VD/EB1089 inhibition of thyroid carcinoma cell growth by p27 hypophosphorylation, diminished association with Skp2, and consequent accumulation. This effect can be mediated but is not essentially dependent on the phosphatase PTEN/Akt/PKB pathway. These properties support the potential utility of VD analogs in the treatment of thyroid carcinomas irrespective of their PTEN/pAkt status.

Figures

Figure 1.

Dose response of VD and EB1089 on thyroid cancer cell proliferation as assessed by thymidine incorporation. WRO cells (a) and NPA cells (b) were treated with VD or EB1089 at the concentrations indicated for 72 hours. Both cell lines shows a comparable ∼50% reduction in response to VD at 10−6 mol/L. TPC-1 and FRO cells showed similar responses to both compounds (not shown).

Figure 2.

Effect of VD on MIB-1 labeling of thyroid carcinoma cell lines. a: A representative photomicrograph of NPA cells showing control cells (left) and cells treated with VD 10−6 mol/L (right). b: All cell lines show a similar dose-dependent reduction in MIB-1 labeling with maximum inhibition of 40 to 55%.

Figure 3.

Effect of VD or EB1089 on p27 expression. a: WRO (left) and NPA cells (right) were treated with VD (top) or its analog EB1089 (bottom) at the concentrations indicated. After 72 hours of incubation, equal amounts (50 μg) of cell lysates were subjected to immunoblotting with antibodies to p27 and actin as shown. b: The graph depicts a quantitative densitometric analysis of the p27/actin ratio representing the mean + SEM of three separate experiments, each in triplicate for each treatment. c: Immunohistochemical localization of p27 was performed on pellets of control and VD-treated cells as described above. Control cells (left) show weak nuclear staining of p27. After treatment with VD (right) nuclear staining is increased and no cytoplasmic staining is seen.

Figure 4.

Effect of VD, EB1089, and/or the proteasome inhibitor LLnL on p27 accumulation. a: WRO and NPA cells were treated with either VD, EB1089, or LLnL alone or in combination as detailed under Materials and Methods. Each bar represents the quantitative densitometric analysis of the p27/actin ratio as a mean + SEM of at least three separate wells from each treatment group from three independent experiments. b: Effect of VD or EB1089 treatment on expression of the Skp2 component of the ubiquitin ligase complex. c: Immunoprecipitated p27 from VD- or EB1089-treated WRO cells was immunoblotted with anti-Skp2. The blot reveals reduced association between p27 and Skp2 in the presence of VD or EB1089 consistent with diminished p27 targeting to the proteasome for degradation.

Figure 5.

Effect of VD or EB1089 on p27 phosphorylation. a: In this representative experiment, WRO cells were treated with VD or EB1089 and lysates immunoprecipitated with a p27 antibody and immunoblotted with p27 antibody (top), total phosphothreonine antibody (middle), or pThr-p27 (bottom). Total p27 levels are significantly greater after treatment with VD or EB1089 but in contrast p27 pThr content remained unchanged by these treatments. b: WRO cells were incubated with ortho-32 P with simultaneous labeling and treatment with VD/EB1089 for 8 hours. Equal amounts of lysates were immunoprecipitated with antibodies to p27 or actin (as indicated), separated on SDS-polyacrylamide gel electrophoresis, and autoradiographed.

Figure 6.

The role of PTEN/Akt/PKB pathway in p27 regulation by VD. a: WRO cells were treated with VD or EB1089 in the presence or absence of the phosphatase inhibitor pervanadate (pV) as indicated. Cell lysates were immunoblotted with antibodies to p27 or actin as indicated. Note the effect of phosphatase inhibition on abrogation of VD/EB1089-mediated p27 accumulation consistent with a role for phosphatase action in mediating VD/EB1089 effect on p27. b: WRO and NPA cells were treated with either VD or EB1089 as detailed under Materials and Methods. Note the induction of PTEN expression by VD/EB1089 in WRO but not NPA cells. Blots were stripped and reprobed with an anti-actin antibody. c: Transfection of PTEN results in p27 accumulation 24 hours after PTEN transfection (left) an effect not significantly enhanced when cells were subsequently treated for 24 hours with VD (right). Note the effect of PTEN introduction on p27 accumulation that was minimally enhanced by the subsequent treatment with VD. d: To investigate the effect of the downstream PI3 kinase pathway on p27 accumulation in thyroid carcinomas, WRO and NPA cells were treated with VD, EB1089, or the PI3-kinase inhibitors wortmannin (wort), or LY900402 (LY) alone or in combination as indicated. Lysates were resolved on SDS-polyacrylamide gel electrophoresis and probed with specific antisera to pAkt/PKB, total Akt/PKB, or p27. Blots were stripped and reprobed with anti-actin antibody. Note the effect of VD/EB1089 on reduction of pAkt/PKB in WRO but not NPA cells consistent with the induction of PTEN activity by VD/EB in WRO but not NPA cells. Note, however, the comparable effects of PI-3 kinase inhibitors on pAkt/PKB and p27 in WRO and NPA cells highlighting the importance of this pathway in p27 regulation in thyroid cancer cells. The results are representative of three separate experiments each of which included at least three separate wells pooled for each treatment group.