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Differential expression of cell cycle regulators in CDK5-dependent medullary thyroid carcinoma tumorigenesis.

Pozo K, Hillmann A, Augustyn A, Plattner F, Hai T, Singh T, Ramezani S, Sun X, Pfragner R, Minna JD, Cote GJ, Chen H, Bibb JA, Nwariaku FE - Oncotarget (2015)

Bottom Line: Finally, the same set of cell cycle proteins was consistently overexpressed in human sporadic MTC but not in hereditary MTC.Together these findings suggest that aberrant CDK5 activity precedes cell cycle initiation and thus may function as a tumor-promoting factor facilitating cell cycle protein expression in MTC.Targeting aberrant CDK5 or its downstream effectors may be a strategy to halt MTC tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry, The University of Texas Southwestern Medical Center, Dallas, TX, USA.

ABSTRACT
Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer of thyroid C-cells, for which few treatment options are available. We have recently reported a role for cyclin-dependent kinase 5 (CDK5) in MTC pathogenesis. We have generated a mouse model, in which MTC proliferation is induced upon conditional overexpression of the CDK5 activator, p25, in C-cells, and arrested by interrupting p25 overexpression. Here, we identify genes and proteins that are differentially expressed in proliferating versus arrested benign mouse MTC. We find that downstream target genes of the tumor suppressor, retinoblastoma protein, including genes encoding cell cycle regulators such as CDKs, cyclins and CDK inhibitors, are significantly upregulated in malignant mouse tumors in a CDK5-dependent manner. Reducing CDK5 activity in human MTC cells down-regulated these cell cycle regulators suggesting that CDK5 activity is critical for cell cycle progression and MTC proliferation. Finally, the same set of cell cycle proteins was consistently overexpressed in human sporadic MTC but not in hereditary MTC. Together these findings suggest that aberrant CDK5 activity precedes cell cycle initiation and thus may function as a tumor-promoting factor facilitating cell cycle protein expression in MTC. Targeting aberrant CDK5 or its downstream effectors may be a strategy to halt MTC tumorigenesis.

No MeSH data available.


Related in: MedlinePlus

Analysis of cell cycle protein expression in human MTC samplesRepresentative immunoblots of lysates from control thyroid tissue (Co), sporadic (Sp) and hereditary (H) human MTC tumors with antibodies as indicated are shown with quantification. Protein levels were normalized to Coomassie blue (CB) signal. Immunoblots were probed with antibodies to A) CDK2, CDK4 and CDK6; B) cyclins-A1, -D1, -E2; C) in p15INK4b, p16INK4a, p18INK4c, p19INK4d, p21CIP/WAF1 and p27KIP. P-values were for CDK2, Sp, p = 0.0168 and H, p = 0.0140; for CDK4, Sp, p = 0.4072 and H, p = 0.8576; for CDK6, Sp, p = 0.5484 and H, p = 0.4916; for cyclin-A1, Sp, p = 0.9923 and H, p = 0.7017; for cyclin-D1, Sp, p = 0.0307 and H, p = 0.6883; for cyclin-E2, Sp, p = 0.2645 and H, p = 0.3070; for p15INK4b, Sp, p = 0.0088 and H, p = 0.0310; for p16INK4a, Sp, p = 0.0014 and H, p = 0.0496; for p18INK4c, Sp, p = 0.0309 and H, p = 0.1054; for p19INK4d, Sp, p = 0.0484 and H, p = 0.3560; for p21CIP/WAF, Sp, p = 0.0554 and H, p = 0.6807; for p27KIP1, Sp, p = 0.2149 and H, p = 0.2131. Data are represented as mean +/− SEM, N = 4 for each condition.
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Figure 5: Analysis of cell cycle protein expression in human MTC samplesRepresentative immunoblots of lysates from control thyroid tissue (Co), sporadic (Sp) and hereditary (H) human MTC tumors with antibodies as indicated are shown with quantification. Protein levels were normalized to Coomassie blue (CB) signal. Immunoblots were probed with antibodies to A) CDK2, CDK4 and CDK6; B) cyclins-A1, -D1, -E2; C) in p15INK4b, p16INK4a, p18INK4c, p19INK4d, p21CIP/WAF1 and p27KIP. P-values were for CDK2, Sp, p = 0.0168 and H, p = 0.0140; for CDK4, Sp, p = 0.4072 and H, p = 0.8576; for CDK6, Sp, p = 0.5484 and H, p = 0.4916; for cyclin-A1, Sp, p = 0.9923 and H, p = 0.7017; for cyclin-D1, Sp, p = 0.0307 and H, p = 0.6883; for cyclin-E2, Sp, p = 0.2645 and H, p = 0.3070; for p15INK4b, Sp, p = 0.0088 and H, p = 0.0310; for p16INK4a, Sp, p = 0.0014 and H, p = 0.0496; for p18INK4c, Sp, p = 0.0309 and H, p = 0.1054; for p19INK4d, Sp, p = 0.0484 and H, p = 0.3560; for p21CIP/WAF, Sp, p = 0.0554 and H, p = 0.6807; for p27KIP1, Sp, p = 0.2149 and H, p = 0.2131. Data are represented as mean +/− SEM, N = 4 for each condition.

Mentions: While hereditary MTC is mainly caused by RET proto-oncogene mutations and consequent deregulation of the RET signaling pathway, the molecular basis for sporadic MTC is not well understood. Some sporadic MTC cases harbor mutations in the RET or Ras genes, but others do not. We previously reported that CDK5 was involved in MTC tumorigenesis, and found that high levels of CDK5 and its activators, p35 and p25, occur predominantly in the sporadic compared to the hereditary form of the disease [10]. Having established that CDK5 activity correlates with elevated cell cycle protein expression levels in proliferating mouse MTC and in a human sporadic MTC cell line, we compared protein levels of CDKs, cyclins and CKI in sporadic and hereditary MTC patient tissues (Figure 5). Sporadic specimen did not exhibit RET mutations (see method section). We found that CDK2 levels were elevated in sporadic cases, but decreased in hereditary forms of MTC. In contrast, CDK4 and CDK6 showed no significant changes in any of the groups analyzed (Figure 5A). Cyclin-D1 levels were increased in sporadic MTC, while cyclin-A1 and cyclin-E2 were unchanged in MTC compared to control thyroid samples (Figure 5B). Cyclin-B1 could not be detected in control thyroid or MTC specimens. p15INK4b and p16INK4a expression was increased in both, sporadic and hereditary MTC. The levels of p18INK4c, p19INK4d and p21CIP/WAF1 were elevated in sporadic but not hereditary MTC. Finally p27KIP1 expression was unchanged in MTC samples compared to control samples. Proliferating Cell Nuclear Antigen was expressed at the same level in sporadic and hereditary tumors, thereby confirming that the observed changes were not just a measure of growth fraction (Figure S1B). The changes in cell cycle regulator expression that were observed in sporadic MTC tissues are consistent with those observed in the human MTC cell line and in mouse tumors (Table 1). The results suggest that the MTC mouse model may more accurately model the molecular mechanisms underlying sporadic MTC than hereditary forms. Thus CDK5 may play a more important role in sporadic than familial MTC tumorigenesis.


Differential expression of cell cycle regulators in CDK5-dependent medullary thyroid carcinoma tumorigenesis.

Pozo K, Hillmann A, Augustyn A, Plattner F, Hai T, Singh T, Ramezani S, Sun X, Pfragner R, Minna JD, Cote GJ, Chen H, Bibb JA, Nwariaku FE - Oncotarget (2015)

Analysis of cell cycle protein expression in human MTC samplesRepresentative immunoblots of lysates from control thyroid tissue (Co), sporadic (Sp) and hereditary (H) human MTC tumors with antibodies as indicated are shown with quantification. Protein levels were normalized to Coomassie blue (CB) signal. Immunoblots were probed with antibodies to A) CDK2, CDK4 and CDK6; B) cyclins-A1, -D1, -E2; C) in p15INK4b, p16INK4a, p18INK4c, p19INK4d, p21CIP/WAF1 and p27KIP. P-values were for CDK2, Sp, p = 0.0168 and H, p = 0.0140; for CDK4, Sp, p = 0.4072 and H, p = 0.8576; for CDK6, Sp, p = 0.5484 and H, p = 0.4916; for cyclin-A1, Sp, p = 0.9923 and H, p = 0.7017; for cyclin-D1, Sp, p = 0.0307 and H, p = 0.6883; for cyclin-E2, Sp, p = 0.2645 and H, p = 0.3070; for p15INK4b, Sp, p = 0.0088 and H, p = 0.0310; for p16INK4a, Sp, p = 0.0014 and H, p = 0.0496; for p18INK4c, Sp, p = 0.0309 and H, p = 0.1054; for p19INK4d, Sp, p = 0.0484 and H, p = 0.3560; for p21CIP/WAF, Sp, p = 0.0554 and H, p = 0.6807; for p27KIP1, Sp, p = 0.2149 and H, p = 0.2131. Data are represented as mean +/− SEM, N = 4 for each condition.
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Related In: Results  -  Collection

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Figure 5: Analysis of cell cycle protein expression in human MTC samplesRepresentative immunoblots of lysates from control thyroid tissue (Co), sporadic (Sp) and hereditary (H) human MTC tumors with antibodies as indicated are shown with quantification. Protein levels were normalized to Coomassie blue (CB) signal. Immunoblots were probed with antibodies to A) CDK2, CDK4 and CDK6; B) cyclins-A1, -D1, -E2; C) in p15INK4b, p16INK4a, p18INK4c, p19INK4d, p21CIP/WAF1 and p27KIP. P-values were for CDK2, Sp, p = 0.0168 and H, p = 0.0140; for CDK4, Sp, p = 0.4072 and H, p = 0.8576; for CDK6, Sp, p = 0.5484 and H, p = 0.4916; for cyclin-A1, Sp, p = 0.9923 and H, p = 0.7017; for cyclin-D1, Sp, p = 0.0307 and H, p = 0.6883; for cyclin-E2, Sp, p = 0.2645 and H, p = 0.3070; for p15INK4b, Sp, p = 0.0088 and H, p = 0.0310; for p16INK4a, Sp, p = 0.0014 and H, p = 0.0496; for p18INK4c, Sp, p = 0.0309 and H, p = 0.1054; for p19INK4d, Sp, p = 0.0484 and H, p = 0.3560; for p21CIP/WAF, Sp, p = 0.0554 and H, p = 0.6807; for p27KIP1, Sp, p = 0.2149 and H, p = 0.2131. Data are represented as mean +/− SEM, N = 4 for each condition.
Mentions: While hereditary MTC is mainly caused by RET proto-oncogene mutations and consequent deregulation of the RET signaling pathway, the molecular basis for sporadic MTC is not well understood. Some sporadic MTC cases harbor mutations in the RET or Ras genes, but others do not. We previously reported that CDK5 was involved in MTC tumorigenesis, and found that high levels of CDK5 and its activators, p35 and p25, occur predominantly in the sporadic compared to the hereditary form of the disease [10]. Having established that CDK5 activity correlates with elevated cell cycle protein expression levels in proliferating mouse MTC and in a human sporadic MTC cell line, we compared protein levels of CDKs, cyclins and CKI in sporadic and hereditary MTC patient tissues (Figure 5). Sporadic specimen did not exhibit RET mutations (see method section). We found that CDK2 levels were elevated in sporadic cases, but decreased in hereditary forms of MTC. In contrast, CDK4 and CDK6 showed no significant changes in any of the groups analyzed (Figure 5A). Cyclin-D1 levels were increased in sporadic MTC, while cyclin-A1 and cyclin-E2 were unchanged in MTC compared to control thyroid samples (Figure 5B). Cyclin-B1 could not be detected in control thyroid or MTC specimens. p15INK4b and p16INK4a expression was increased in both, sporadic and hereditary MTC. The levels of p18INK4c, p19INK4d and p21CIP/WAF1 were elevated in sporadic but not hereditary MTC. Finally p27KIP1 expression was unchanged in MTC samples compared to control samples. Proliferating Cell Nuclear Antigen was expressed at the same level in sporadic and hereditary tumors, thereby confirming that the observed changes were not just a measure of growth fraction (Figure S1B). The changes in cell cycle regulator expression that were observed in sporadic MTC tissues are consistent with those observed in the human MTC cell line and in mouse tumors (Table 1). The results suggest that the MTC mouse model may more accurately model the molecular mechanisms underlying sporadic MTC than hereditary forms. Thus CDK5 may play a more important role in sporadic than familial MTC tumorigenesis.

Bottom Line: Finally, the same set of cell cycle proteins was consistently overexpressed in human sporadic MTC but not in hereditary MTC.Together these findings suggest that aberrant CDK5 activity precedes cell cycle initiation and thus may function as a tumor-promoting factor facilitating cell cycle protein expression in MTC.Targeting aberrant CDK5 or its downstream effectors may be a strategy to halt MTC tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry, The University of Texas Southwestern Medical Center, Dallas, TX, USA.

ABSTRACT
Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer of thyroid C-cells, for which few treatment options are available. We have recently reported a role for cyclin-dependent kinase 5 (CDK5) in MTC pathogenesis. We have generated a mouse model, in which MTC proliferation is induced upon conditional overexpression of the CDK5 activator, p25, in C-cells, and arrested by interrupting p25 overexpression. Here, we identify genes and proteins that are differentially expressed in proliferating versus arrested benign mouse MTC. We find that downstream target genes of the tumor suppressor, retinoblastoma protein, including genes encoding cell cycle regulators such as CDKs, cyclins and CDK inhibitors, are significantly upregulated in malignant mouse tumors in a CDK5-dependent manner. Reducing CDK5 activity in human MTC cells down-regulated these cell cycle regulators suggesting that CDK5 activity is critical for cell cycle progression and MTC proliferation. Finally, the same set of cell cycle proteins was consistently overexpressed in human sporadic MTC but not in hereditary MTC. Together these findings suggest that aberrant CDK5 activity precedes cell cycle initiation and thus may function as a tumor-promoting factor facilitating cell cycle protein expression in MTC. Targeting aberrant CDK5 or its downstream effectors may be a strategy to halt MTC tumorigenesis.

No MeSH data available.


Related in: MedlinePlus