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. Author manuscript; available in PMC: 2014 Oct 14.
Published in final edited form as: Cancer Cell. 2013 Oct 14;24(4):10.1016/j.ccr.2013.08.027. doi: 10.1016/j.ccr.2013.08.027

The Role of Cdk5 in Neuroendocrine Thyroid Cancer

Karine Pozo 1,9,*, Emely Castro-Rivera 1,*, Chunfeng Tan 1, Florian Plattner 1, Gert Schwach 2, Veronika Siegl 2, Douglas Meyer 1, Ailan Guo 3, Justin Gundara 4, Gabriel Mettlach 1, Edmond Richer 5, Jonathan A Guevara 6, Li Ning 7, Li-Huei Tsai 8, Xiankai Sun 9, Pietro Antich 5, Stanley Sidhu 4, Bruce G Robinson 4, Herbert Chen 7, Fiemu E Nwariaku 10,11, Roswitha Pfragner 2, James A Richardson 12,13, James A Bibb 1,11,14
PMCID: PMC3849320  NIHMSID: NIHMS521609  PMID: 24135281

SUMMARY

Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer that originates from calcitonin-secreting parafollicular cells, or C cells. We found that Cdk5 and its cofactors, p35 and p25, are highly expressed in human MTC and that Cdk5 activity promotes MTC proliferation. A conditional MTC mouse model was generated and corroborated the role of aberrant Cdk5 activation in MTC. C cell-specific overexpression of p25 caused rapid C cell hyperplasia leading to lethal MTC, which was arrested by repressing p25 overexpression. A comparative phosphoproteomic screen between proliferating and arrested MTC identified the retinoblastoma protein (Rb) as a crucial Cdk5 downstream target. Prevention of Rb phosphorylation at Ser807/811 attenuated MTC proliferation. These findings implicate Cdk5 signaling via Rb as critical to MTC tumorigenesis and progression.

Keywords: p25/Cdk5, medullary thyroid carcinoma, tumorigenesis, MEN2A

INTRODUCTION

Neuroendocrine tumors are relatively rare neoplasms characterized by abnormal hormone secretion, an indolent course, specific genetic mutations, and a poor response to conventional therapies. MTC arises from C cells and metastasizes frequently to regional lymph nodes, bones, lungs, liver and brain. Although MTC accounts for only 3 – 5% of all thyroid cancers, it represents over 14% of thyroid cancer-related deaths and affects both men and women, almost equally (Massoll and Mazzaferri, 2004; Sippel et al., 2008).

Approximately 25% of MTC cases are hereditary and occur as familial MTC or as a component of the multiple endocrine neoplasia 2 (MEN2) syndromes. MEN2A and MEN2B are autosomal dominant syndromes, in which MTC is frequently associated with pheochromocytoma (adrenal gland cancer). MEN2B is the more severe form of the syndromes and affected patients develop additional symptoms such as mucosal neuromas and marfanoid habitus. These genetic forms of MTC originate from activating germline mutations in the RET (Rearranged during transfection) proto-oncogene (Liska et al., 2005), which encodes a tyrosine kinase receptor for the glial-derived neurotrophic factor family (GDNF, neurturin, artemin and persephin) (Takahashi, 2001). Mechanisms by which RET mutations lead to MTC have been extensively studied (Asai et al., 2006). However, the majority of MTC cases (>75%) arise spontaneously and only 40% of these so-called sporadic cases are caused by somatic RET mutations.

The molecular basis of sporadic MTC is poorly understood (Hu and Cote, 2012), although mutations in the RAS family of small GTPases genes have recently been identified in RET-negative MTC cases and RAS has been proposed to act as an alternative driver to RET in MTC tumorigenesis (Agrawal et al., 2013; Boichard et al., 2012; Ciampi et al., 2013). Consequently, therapeutic strategies are limited (Schlumberger et al., 2008). Currently, complete surgical removal of the thyroid remains the primary treatment for early stage MTC patients (Fialkowski and Moley, 2006). However, recurrence is common and metastasis are resistant to chemotherapy. The recent FDA-approval of the tyrosine-kinase inhibitor, Vandetanib, provides a treatment option for MTC (Wells et al., 2012). However the use of this drug is limited to unresectable, late stage, and metastatic MTC and may produce unwanted side effects (Sherman, 2013). Thus, additional therapies are needed and identifying novel molecular mechanisms underlying MTC tumorigenesis is crucial for the development of treatments for MTC.

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase essential for the central nervous system development and brain function (Angelo et al., 2003). Binding with its non-cyclin cofactors, p35 or p39, activates Cdk5. Cellular stress can induce the cleavage of p35 by the Ca2+-dependent protease calpain to p25, which aberrantly activates Cdk5 thereby promoting the phosphorylation of substrates implicated in neurodegeneration (Kusakawa et al., 2000). Cdk5’s functions beyond the nervous system are still emerging. It modulates insulin secretion in pancreatic β cells (Lilja et al., 2001; Wei et al., 2005) and may contribute to cell cycle regulation and some forms of cancer (Goodyear and Sharma, 2007; Jiang et al., 2005; Kim et al., 2006; Lin et al., 2007; Lopes and Agostinho, 2011; Strock et al., 2006). Indeed, elevated Cdk5 activity has been detected in pancreatic and lung cancers (Demelash et al., 2012; Feldmann et al., 2010). Furthermore, Cdk5 regulates motility and migration of a variety of cancer cell lines, which suggests a role for Cdk5 in tumor progression and metastasis (Huang et al., 2009; Quintavalle et al., 2011; Strock et al., 2006).

Neuroendocrine and neuronal cells both originate from the neural crest and share common physiological features (Pang and Sudhof, 2010). Given that Cdk5 plays a crucial role in neuronal physiology, we hypothesized that Cdk5 also regulates neuroendocrine cell function. Here we investigate the role of Cdk5 in human MTC tumorigenesis.

RESULTS

The role of Cdk5 and its activators, p35 and p25, in human MTC proliferation

We detected Cdk5 in human thyroid gland and specifically in neuroendocrine C cells as shown by colocalization of Cdk5 with calcitonin (Figures 1A and 1B). Expression of p35/p25 was also evident in both follicular and parafollicular C cells as detected by immunostaining contiguous thyroid sections with a p35 antibody, which also detects p25 (Figure 1A; Figures S1A and S1B). As neoplastic C cell hyperplasia is an early step in the development of MTC, we examined Cdk5 and p35/p25 expression in resected malignant specimens from 17 MTC patients that underwent thyroidectomy. Cdk5 and p35/p25 expression was apparent in all MTC samples (Figures 1A, 1C, and 1D; Figure S1A). Notably, Cdk5 and its activators were expressed at higher levels in sporadic MTC samples than in non-cancerous thyroid tissues (Figures 1C and 1D; Figures S1A). Interestingly, Cdk5, p35, and p25 levels were not consistently elevated in MEN2A specimens compared to control tissues, suggesting possible mechanistic differences between at least some hereditary and sporadic forms of this cancer (Figure 1D). Furthermore, rising levels of MTC (% neoplastic cellularity) correlated with increasing p35/p25 expression in sporadic MTC patient samples that were graded histopathologically (Figure 1E). Altogether, these findings suggest that Cdk5 activity may be important for human sporadic MTC tumorigenesis.

Figure 1. Cdk5 and its cofactors are expressed in human thyroid and in MTC.

Figure 1

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(A) Contiguous sections of normal human thyroid, sporadic medullary thyroid tumors, and hereditary medullary thyroid tumors (MEN2A syndrome) are immunostained for calcitonin, Cdk5, and p35/25. Scale bars are 20 μm for normal tissue stains and 100 μm for tumor stains (MEN2A, sporadic).

(B) High magnification image of normal thyroid costained for Cdk5 (brown) and calcitonin (dark blue). The arrow indicates C cell. Scale bar, 20 μm.

(C) Representative immunoblots of lysates from non-malignant (Norm), sporadic (Spo), and familial (MEN2A) cancerous human thyroid specimens for Cdk5, p35/p25 and GAPDH are shown.

(D) Quantification of (C). Each point represents the protein expression level normalized to the loading control for each sample. Horizontal bar lines represent mean values for non-malignant (n = 4 – 8), sporadic (n = 10 – 12), and MEN2A (n = 7). For Cdk5, Spo, p = 0.0048 and MEN2A, p = 0.4411; for p35, Spo, p = 0.0317 and MEN2A, p = 0.0793; for p25, Spo, p < 0.0001 and MEN2A, p = 0.2305 in a two-tailed unpaired Student’s t-tests with Welch’s correction. Vertical error bars represent S.E.M.

(E) Immunoblots showing the expression of Cdk5, p35, p25 in samples of sporadic MTC patients at different stages of the disease and quantification. The percentage of MTC cellularity was determined during the pathological analysis by quantifying microscopically the percentage of a representative slice that was occupied by the MTC (see Supplemental Methods). Values for each MTC sample were normalized to the loading control. Data represents mean values of n = 1 – 8 samples as indicated. Error bars represent S.E.M.

See also Figure S1.

To investigate further the possible role of Cdk5 in MTC progression, we assessed Cdk5, p35, and p25 expression in a panel of 6 human cell lines derived from sporadic MTC (Pfragner et al., 2002). All MTC cell lines exhibited Cdk5 and p35 expression as well as p25 generation (Figure 2A). Because cultures of normal human thyroid C cell cultures are difficult to establish due to the low C cell content in a human thyroid biopsy (~ 1%), cultured normal human diploid fibroblasts (NDF) were used as controls. NDF also exhibited p35 expression. However, very little Cdk5 or p25 were detected in these cells. Treatment of MTC cells with the Cdk5 inhibitor CP681301 (Karran and Palmer, 2007; Sadleir and Vassar, 2012; Wen et al., 2008) stopped their proliferation and significantly reduced their viability (Figures 2B and S2A). However, Cdk5 inhibition had little or no effect on the proliferation and viability of NDF cells (Figure S2A). With these positive results in hand, MTC-SK and SIN-J were selected as representative for further analysis. MTC-SK cells were derived from a solid sporadic MTC tumor while SIN-J cells originated from metastatic sporadic MTC (Pfragner et al., 1990; Pfragner et al., 1993). Both have been characterized for their close similarity to MTC tumor cells and express appreciable levels of Cdk5, p35, and p25.

Figure 2. MTC cell proliferation is dependent upon Cdk5 activity.

Figure 2

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(A) Immunoblots of lysates from the indicated human sporadic MTC cell lines and NDF probed with antibodies to Cdk5, p35, p25 and GAPDH.

(B) Dose-dependent effect CP681301 on MTC-SK cell proliferation and viability.

(C) Effect of CP681301 on Cdk5 and Cdk2 activity. Cdk5 and Cdk2 were immunopurified from MTC-SK cells treated with 0, 1 or 5 μM CP681301 for 12 hr and used to phosphorylate histone H1 in vitro. Immunoblots show Cdk5 and Cdk2 used in immunoprecipitation-kinase assay reaction mixtures (top). Kinase activity was detected by blotting for phospho-histone H1 (p-H1) (middle). Coomassie stained (CB) histone H1 controls show the amount of substrate used in reactions (bottom). For quantitation of p-H1, for Cdk5 activity, p = 0.0037 for 0 versus 1 μM; p = 0.030 for 0 versus 5 μM.

(D) Immunoblots of lysates from MTC-SK cells transfected with a control plasmid (Con) or with a KD-Cdk5 expression vector (KD) probed with antibodies to Cdk5 and GAPDH (top panel); an histogram summarizing the effect of Con or KD expression on cell proliferation (p = 0.0057) (bottom panel).

(E) Immunoblots for p-H1 show Cdk5 and Cdk2 activity in MTC-SK cells transfected with a control plasmid (Con) or with KD-Cdk5 (KD) (for Cdk5 activity, p = 0.0297). Immunoprecipitated Cdk5, Cdk2 and Coomassie stained histone H1 controls are shown.

(F) Immunoblots of lysates from MTC-SK cells transfected with a scrambled siRNA (Con) or a p35 siRNA probed with antibodies to p35 and GAPDH (top panel); an histogram summarizing the effect of Con or p35 siRNA expression on cell proliferation (p = 0.0365) (bottom panel).

(G) Immunoblots for p-H1 show Cdk5 and Cdk2 activity in MTC-SK cells transfected with scrambled (Con) or p35 siRNA (for Cdk5 activity, p = 0.0178). Immunoprecipitated Cdk5, Cdk2 and Coomassie stained histone H1 controls are shown.

All data represent mean values, error bars are S.E.M and p-values were determined by a two-tailed paired Student’s t-test, n = 4 (C-G). For B, D, and G, number of cells is 1000-fold. See also Figure S2.

CP681301 is moderately selective for Cdk5 over Cdk2 in vitro (2.8-fold). To confirm CP681301 specificity for Cdk5 over Cdk2, we immunoprecipitated either Cdk5 or Cdk2 using cell lysates from CP681301-treated MTC-SK and SIN-J cells and assessed the activity of each kinase in vitro using histone H1 as a substrate. Cdk5 but not Cdk2 activity was inhibited by CP681301 at the concentrations used in this study for both cell lines (Figure 2C; Figure S2B). Furthermore, competing with endogenous Cdk5 activity by expressing a kinase-dead (KD) Cdk5 mutant also reduced MTC-SK (Figure 2D) and SIN-J (Figure S2C) cell proliferation. As for pharmacological inhibition, this reduction in cell growth correlated with a reduction in Cdk5 activity in both cell lines (Figure 2E; Figure S2D). Moreover, siRNA-mediated knockdown of the Cdk5 activator p35 also reduced MTC-SK and SIN-J cell growth (Figure 2F; Figure S2E). Again, p35 siRNA significantly reduced Cdk5 activity in MTC-SK and SIN-J cells without affecting Cdk2 activity (Figure 2G; Figure S2F). Together these findings indicate that Cdk5 activity, as dictated by the expression of its activating cofactors p35 and p25, is critical to the proliferation of human sporadic MTC cells and may contribute to some cases of MTC.

P25 overexpression and associated Cdk5 activity result in thyroid tumor formation

To delineate the effects of aberrant Cdk5 activation in MTC tumorigenesis, we used the neuron specific enolase (NSE) promoter to drive GFP-tagged p25 overexpression (p25OE) in C cells. In this bitransgenic mouse model, p25OE is regulated via a tetracycline-controlled transactivator (tTA) system, which is driven by the NSE promoter (Figure 3A) (Meyer et al., 2008). In this model, p25OE can be induced by withdrawal of the tetracycline-analogue, doxycycline, from the animal diet (Cruz et al., 2003), while administration of doxycycline readily represses p25OE. The p25OE mice developed large, bilateral thyroid tumors 16 – 25 weeks after doxycycline removal (Figure 3B). These fast growing tumors were malignant as shown by FDG uptake during PET imaging (Figures 3C and 3D). No p25OE mice survived past 30 weeks off doxycycline (Figure 3E). In contrast, control littermates that lacked p25-GFP transgene had normal thyroids and survival rates (Figures 3B and 3E). The tumors expressed high levels of p25-GFP (Figures 3F and 3G) and were formed by calcitonin-positive neoplastic C cells that invaded the tracheal muscle and extended into the tracheal lumen leading to obstruction (Figures 3H and 3I). Clusters of calcitonin-positive C cells invaded and metastasized within the vasculature in the skeletal muscles near the lungs (Figure 3J). Together these histological analyses indicate that these thyroid tumors are MTC.

Figure 3. Characterization of a MTC animal model.

Figure 3

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(A) Schematic of the bitransgenic system showing activation of TetOp promoter-controlled p25-GFP expression by doxycycline (Dox)-inhibitable tTa. tTA expression is under NSE promoter control.

(B) Dissection showing the thyroid gland in p25OE and. control littermate. The arrow indicates tumors. L, larynx; T, trachea.

(C) In vivo microPET/CT of WT thyroid vs. p25OE thyroid tumor (arrows). Representative saggital (top), coronal (middle), and transaxial (bottom) images are shown.

(D) Time-dependent [18F]FDG uptake in thyroid (WT) and thyroid tumors (p25OE). Mean values are plotted. Error bars represent S.E.M (n = 3).

(E) Survival curve for p25OE and control littermates (n = 28).

(F) Optical fluorescent imaging of thyroid tumors (arrow) in a p25OE mouse.

(G) Immunoblots of thyroid (littermate control, Con) versus p25OE thyroid tumor lysates for p25-GFP (using anti-GFP antibodies) and GAPDH.

(H) Calcitonin staining (left, scale bar, 100 μm) and GFP immunostaining (right, scale bar, 500 μm) of a p25OE tumor.

(I) Representative hematoxylin and eosin (HE) stainings (top panels) and corresponding calcitonin immunostains (bottom panels) of the thyroid and tracheal region in a p25OE mouse (scale bars are 500 μm, top left, 200 μm, top and bottom right, 100 μm, bottom left). T, Trachea; MTC, medullary thyroid carcinoma.

(J) HE staining of metastatic C cells in skeletal muscle vasculature (left, scale bar, 20 μm), and HE staining (middle) and calcitonin immunostain (right) of metastatic C cells within alveolar walls of lung (scale bar 50 μm) of contiguous sections.

To monitor MTC tumorigenesis, thyroid samples were examined at different time points following induction of p25OE (Figure 4A). In control animals, thyroid gland was normal, comprised of follicular cells surrounding distinct colloid-filled follicles. C cells were interspersed in-between follicular cells as detected by a calcitonin-specific immunostaining (Figure S3). After 5 weeks of p25OE, mild C cell hyperplasia developed and progressed into small tumors within 11 weeks. By 16 weeks, large bilateral MTC tumors had formed and invaded the space between trachea and esophagus. At this stage, no more follicles were visible. The thyroid tumors were divided by fibrous septa in a nested pattern and consisted of a population of round cells with uniform nuclei and amphophilic cytoplasm, characteristic of MTC. All mice examined died within 30 weeks of p25OE. However, stopping p25OE after 5, 11, or 16 weeks led to 100% survival of mice analyzed for up to 32 weeks (Figure 4B). Indeed, switching-off p25OE at early stages of MTC tumorigenesis (5 weeks) prevented C cell hyperplasia, while repressing p25OE at later stages of the disease (11, 16 weeks) resulted in the arrest of tumor growth (Figure 4C). As expected, arrested tumors lacked p25-GFP expression. However, repressing p25OE did not affect Cdk5 or p35 protein levels (Figure 4D). Cdk5 activity was significantly reduced in arrested (p25OE Off) tumors compared to proliferating (p25OE On) tumors in which p25 aberrantly activates Cdk5 (Figure 4E).

Figure 4. Progression and arrest of MTC in p25OE mice is controlled by p25 expression and associated aberrant Cdk5 activity.

Figure 4

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(A) HE staining of thyroid in control mice and mice with p25OE On for the indicated time period. Scale bars are 100 μm.

(B) Schematic of p25OE On and Off with survival for each paradigm indicated (n = 4 – 7).

(C) Representative HE staining of typical thyroid for each paradigm of p25OE On followed by arrest (p25OE Off). Scale bars are 100 μm.

(D) Immunoblots of lysates from proliferating (p25OE On, 16 weeks) and arrested (p25OE On, 16 weeks, followed by p25 Off, 6 weeks) tumors (n = 4) for GFP-tagged p25, p35, Cdk5, and GAPDH are shown.

(E) Immunoblot for p-H1 showing Cdk5 activity in proliferating (− Dox) and arrested (+ Dox) tumors as determined by immunoprecipitation/kinase assays using histone H1 as a substrate. Inhibition of Cdk5 by indolinone A, immunoprecipitated Cdk5, and Coomassie stained (CB) histone H1 controls are shown.

See also Figure S3

Together these results demonstrate that NSE promoter-driven p25OE results in the formation of thyroid tumors featuring the characteristics of human MTC (Fialkowski and Moley, 2006) and that aberrant Cdk5 activation by p25-GFP contributes to MTC proliferation in this mouse model.

In vitro characterization of p25OE mice MTC

To further confirm the role of Cdk5 in MTC, we generated a cell line from the MTC of p25OE mice (MTCp25). These cells grew as floating clusters and exhibited anchorage-independent growth and malignant transformation in a soft agar assay (Figure 5A). Importantly, MTCp25 cells retained the characteristics of the mouse tumors. These cells were calcitonin-positive, expressed p25-GFP and Cdk5 (Figure 5B), and secreted calcitonin in the culture media (approx. 100 pg/ml). As shown in the tumors p25-GFP expression and Cdk5 activity were controlled by doxycycline (Figures 5C and 5D). Repressing p25-GFP overexpression by culturing the cells in the presence of doxycycline dose-dependently arrested cell proliferation but had no effect on cell viability (Figure 5E). Furthermore, doxycycline had no effect on the proliferation of a heterologous non-small lung cell carcinoma line, H1299 (Figure S4A). Thus turning off p25OE with doxycycline stops mouse MTCp25 cell growth without causing cell death.

Figure 5. Generation of a mouse MTC cell line (MTCp25) that retains the characteristics of p25OE mice tumors.

Figure 5

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(A) Brightfield and epifluorescent images showing MTCp25 mouse cells growing in suspension as cell clusters and colony formation in soft agar.

(B) Immunostaining of MTCp25 cells for Cdk5 (magenta), p25-GFP (yellow), calcitonin (gray), DAPI-stained nucleus (blue), scale bar, 20 μm.

(C) Immunoblots of lysates from doxycycline-treated MTCp25 cells for p25 and Cdk5 showing the effect of doxycycline on p25-GFP overexpression and on Cdk5 expression.

(D) Immunoblot for p-H1 showing the effect of doxycycline on Cdk5 activity after immunoprecipitating Cdk5 from MTCp25 cells treated with doxycycline. Cdk5 inhibition by indolinone A, immunoprecipitated Cdk5 and Coomassie stained (CB) histone H1 controls are shown.

(E, F) Dose-dependent effect of doxycyline (E) and CP681301 (F) on MTCp25 cell proliferation and viability.

(G) Immunoblots of lysates from MTCp25 cells transfected with a scrambled siRNA (Con) or a Cdk5 siRNA (siRNA) probed with antibodies to Cdk5 and GAPDH (top panel); histogram summarizes the effect of Con or Cdk5 siRNA expression on cell proliferation (p = 0.023) (bottom panel).

(H) Immunoblot for p-H1 showing immunoprecipitated Cdk5 activity in cells transfected with Cdk5 siRNA and quantification (p = 0.0145). immunoprecipitated Cdk5, and Coomassie stained (CB) histone H1 controls are shown.

All data represents mean values (n = 4) and error bars are S.E.M; p-values were determined by a two-tailed paired Student’s t-test. For E, F, G, cell numbers are 1000-fold. See also Figure S4

To substantiate the role of Cdk5 activity on MTC tumorigenesis, the effect of Cdk5 inhibition on MTCp25 cell proliferation and viability was assessed. CP681301 dose-dependently reduced MTCp25 cell proliferation and viability as was observed with human MTC cell lines (Figure 5F). Moreover, two alternate Cdk5 inhibitors, indolinone A and roscovitine, also stopped MTCp25 cell proliferation (Figures S4B and S4C). Finally, siRNA knockdown of Cdk5 reduced Cdk5 expression and activity as well as cell growth (Figures 5G and 5H). Taken together, these findings show that MTCp25 cell proliferation is dependent on the Cdk5 activity that is mediated by p25OE, further supporting a role for Cdk5 in MTC progression.

Retinoblastoma protein plays a critical role downstream of Cdk5 activation in MTC proliferation

To identify Cdk5 downstream effectors, we profiled proteins containing Cdk5 consensus phosphorylation sites in proliferating versus arrested p25OE mouse tumors by phospho-scan liquid chromatography tandem mass-spectrometry (Rush et al., 2005). Peptides phosphorylated at serine 807 of Rb were highly enriched in proliferating mouse tumors compared with arrested tumors (Figure S5A), which was confirmed by immunoblotting (Figure 6A). Likewise, phospho-Ser807/811 Rb levels were increased in doxycycline-deprived cultured MTCp25 cells in which p25OE is induced and Cdk5 activity is elevated (Figure 6B). Furthermore, consistent with Cdk5, p35 and p25 expression, phospho-Ser807/811 Rb was elevated significantly in human sporadic MTC tumors but not MEN2A samples compared to control tissue (Figure 6C). Together, these results suggest a mechanistic link between Cdk5 activity, Rb phosphorylation, and sporadic MTC.

Figure 6. Rb is a downstream target of Cdk5 in MTC.

Figure 6

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(A) Immunoblots of lysates from proliferating (p25OE On) and arrested (p25OE Off) p25OE tumors probed with antibodies to phospho-Ser807/811 (pS-Rb) and total Rb (n = 4).

(B) Effect of doxycycline-induced repression on pS-Rb levels in MTCp25 cells.

(C) Immunoblots of lysates from normal thyroid (Norm) (n = 5), sporadic (Spo) (n = 6) and hereditary (MEN2A) (n = 7) MTC patient specimens were probed with antibodies to pS-Rb and GAPDH. For Spo, p = 0.0117; MEN2A, p = 0.1316, two-tailed unpaired Student’s t-test with Welch’s correction.

(D, E) Immunoblots of lysates from MTCp25 cells (D) and MTC-SK cells (E) following a 16 hr CP681301 treatment probed with antibodies to pS-Rb and GAPDH and quantification.

(F, G) Immunoblots from lysates of MTC-SK cells transfected with (F) a control plasmid (Con) or a kinase-dead Cdk5 expression vector (KD) and (G) with a scrambled siRNA (Con) or a p35 siRNA (siRNA) and probed with antibodies to pS-Rb and GAPDH and quantification (in (F) p = 0.0057; in (G) p = 0.0365).

(H) Immunoblots showing the effect of SIP on the phosphorylation of endogenous Rb (at Ser807/811) and STAT3 (left panel). Effect of 10 μM Rb SIP on MTCp25 (left) and MTC-SK (right) cell proliferation are shown (right panel). For MTCp25, p = 0.0336; MTC-SK, p = 0.0157.

(I) Immunoblots of lysates from proliferating (p25OE On) and arrested (p25OE Off) mouse tumors probed with antibodies to pS-Rb, Cdk2, Cyclin A and GAPDH.

(J) Immunoblots of lysates from MTC-SK cells treated with CP681301 (5 μM) for 0, 12, 24 hr probed with antibodies to pS-Rb, Cdk2, Cyclin A, Cdk5 and GAPDH and quantification. For pS-Rb, p < 0.0001 for 0 versus 12 hr, p < 0.0002 for 0 versus 24 hr; for Cdk2, p = 0.0315 for 0 versus 12 hr, p = 0.0104 for 0 versus 24 hr; for Cyclin A, p = 0.0257 for 0 versus 24 hr.

(K) Effect of CVT-313 on sporadic MTC-SK cell proliferation and viability.

(L) Immunoblots of lysates from MTC-SK cells treated with CVT-313 for 12–14 hr probed with antibodies to pS-Rb, Cdk5 and GAPDH.

(M) Immunoblot for p-H1 showing Cdk2 and Cdk5 activity in MTC-SK cells treated with CVT-313 for 12–14 hr. For Cdk2, p = 0.0051 for 0 versus 25 μM.

(N) A schematic model for the Cdk5-Rb-Cdk2 pathway in MTC. Rb binds to E2F and suppresses its transcriptional activity thereby preventing cell proliferation. Upon Rb phosphorylation by Cdk5, E2F is released and activates the transcription of target genes including Cdk2 and Cyclin A that mediate cell proliferation.

All data are mean values (n = 4) and error bars represent SEM; p-values were determined by a two-tailed paired Student’s t-test. For H, K, cell numbers are 1000-fold. See also Figure S5.

Since Ser807/811 are known Cdk5 phosphorylation sites (Futatsugi et al., 2012; Hamdane et al., 2005) and Rb deletion has been linked to neuroendocrine tumorigenesis (Takahashi et al., 2006; Williams et al., 1994), we postulated that Rb is a downstream effector of Cdk5 and that Cdk5-Rb signaling promotes MTC. Indeed, inhibiting Cdk5 activity in mouse and human MTC cells by treatment with CP681301 decreased phospho-Ser807/811 Rb in a dose-dependent manner and stopped cell growth (Figures 6D and 6E; Figures S5B; Figure 2B; Figure S2A). Furthermore, disrupting Cdk5 activity in human MTC cells by expressing a kinase-dead Cdk5 or knock-down of p35 diminished phospho-Ser807/811 Rb and reduced MTC cell proliferation (Figures 6F and 6G; Figures 2D and 2F; Figures S5C, S5D, S2C and S2E). Finally, competing with Cdk5 phosphorylation of Rb by treating mouse or human MTC cell lines with a small inhibiting peptide (SIP) that consisted of a 19 amino acid Rb sequence encompassing Ser807/811 dose-dependently reduced phospho-Ser807/811 Rb and cell proliferation (Figure 6H; Figure S5E). The SIP had no effect on STAT3 phosphorylation at known Cdk5 sites indicating specificity for Rb phosphorylation (Figure 6H). Together, these findings identify Rb as a Cdk5 downstream target in MTC tumorigenesis.

Having confirmed Rb as an important Cdk5 target, we investigated the mechanism by which Cdk5-Rb mediate MTC proliferation. Rb acts as a tumor suppressor by binding the E2F transcription factors, thereby blocking their transcriptional activity. Upon Rb phosphorylation by cyclin-dependent kinases, E2Fs are released and activate the expression of their target genes, including Cdk2 and Cyclin A (Chellappan S.P., 1991; DeGregori et al., 1997; Knudsen et al., 1999). In agreement with this, arrest of mouse tumor growth by repressing p25OE abolished Cdk2 and Cyclin A expression (Figure 6I). Furthermore, inhibiting Cdk5 activity in human MTC cells with CP681301 attenuated Cdk2 and Cyclin A expression in MTC-SK cells by 25% within 12 hr and by 40% after 24 hr of treatment (Figure 6J). In SIN-J cells, CP681301 reduced Cdk2 and Cyclin A levels by more than 50% within 12 hr (Figure S5F). As expected, Rb phosphorylation levels were reduced by more than 50% within 12 hr following CP681301 application to both cell lines. Thus, Cdk2 and Cyclin A appear to be invoked as downstream effectors of the Cdk5-Rb signaling and this cascade may mediate MTC tumorigenesis. In line with these observations, MTC cell growth was reduced by pharmacological blockade of Cdk2 activity with CVT-313 (Figure 6K and Figure S5G). Importantly, inhibition of Cdk2 only partially reduced Rb phosphorylation but had no effect on Cdk5 expression and activity (Figures 6L and 6M; Figure S5H). This effect is consistent with the ability of Cdk2 to contribute to Rb phosphorylation and cell cycle progression (Harbour et al., 1999). Thus, Cdk5 may contribute meaningfully to the progression of some forms of MTC by inactivating Rb and enabling the expression of the E2F target genes, Cdk2 and Cyclin A (Figure 6N).

DISCUSSION

While tremendous advances have been made in understanding how RET causes MTC, little is known about other mechanisms that contribute to the majority of neuroendocrine thyroid cancers. Here, we demonstrate that Cdk5 is crucial for human MTC cell proliferation and thus it contributes to MTC progression. Previous work implicated Cdk5/p35 in the proliferation of the TT cell line, which was derived from familial human MTC and contains a RET mutation at codon 634 resulting in constitutive RET activation (Berger et al., 1984; Lin et al., 2007). It was suggested that p35, but not p25, drives Cdk5 activity and is necessary to maintain the RET-dependent growth of this cell line, implying that Cdk5 activation may be downstream of RET. However we found that dysregulation of Cdk5 activity by overexpressing p25 caused MTC, thus suggesting that Cdk5 rather than RET may trigger MTC. Nevertheless, a link between Cdk5 activity in human MTC and the RET or RAS signaling pathways cannot be ruled out and should be further explored.

In evaluating pro-proliferative downstream effectors for Cdk5, we found that Cdk5 phosphorylates Rb and that inactivation of Cdk5 prevents Rb phosphorylation and reduces Cdk2 and Cyclin A expression in human MTC cells and arrested mouse tumors. Deregulation of Rb signaling is a well-known cause of cancer (Weinberg, 1995). Rb is a tumor suppressor that prevents cell cycle progression from G1 to S by binding and sequestering E2F transcription factors (Sellers et al., 1995). Upon Rb phosphorylation by cyclin/cyclin-dependent kinase complexes, E2Fs are released and activate the transcription of target genes whose products are necessary for cell cycle progression (Bracken et al., 2004). Numerous mitogenic and oncogenic pathways invoke Cyclin D expression. The resulting Cyclin D-Cdk4/6 complexes catalyze the initial Rb phosphorylation while Cyclin A-Cdk2 can contribute to the maintenance of the phospho-Rb-dependent neoplastic state (Nevins, 2001). Our findings raise the possibility that Cdk5 may catalyze the initial Rb phosphorylation and replace Cyclin D-Cdk4/6 complexes in some neuroendocrine cancers.

In support of this hypothesis, here Rb phosphorylation at Ser807/811 correlated with Cdk5 activity in growing mouse tumors and in human MTC cells and was arrested in MTC cells overexpressing dominant negative, kinase-dead Cdk5 or subjected to p35 knock-down. Previously, Ser807/811 Rb was reported as a substrate of aberrant Cdk5 in injured neurons (Panickar et al., 2008). The present results implicate Rb as a likely Cdk5 substrate in MTC tumorigenesis. Given that Cdk5 plays important roles in the central nervous system, using a Cdk5 inhibitor to treat MTC may have undesirable side effects. Targeting protein-protein interactions such as those of Rb or other pro-proliferative downstream effectors may provide more selectivity for eventual clinical applications.

Previously, loss of Rb and E2F transcription factors alleles has been linked to neuroendocrine cancers including MTC (Salon et al., 2007; Takahashi et al., 2004; Ziebold et al., 2003). For example, mice carrying a single functional copy of the Rb wild-type gene develop MTC and this tendency is increased in the C57BL6 mouse strain (Harrison et al., 1995; Leung et al., 2004; Nikitin et al., 1999; Williams et al., 1994; Yamasaki et al., 1998). In humans, genetic analysis of sporadic and hereditary MTC patients have identified mutations in genes encoding the INK4 and CIP/KIP families of CDK inhibitors, that are negative regulators of the Rb pathway. Specifically, somatic mutations in the CDKN2C gene (p18INK4 gene) have been found in several studies and correlated to higher MTC proliferation rates (Flicker et al., 2012; van Veelen et al., 2009). Additionally, mutations within chromosome 19p13.2, which contains the CDKN2D gene (p19INK4D gene), have also been detected frequently in MTC patients (Flicker et al., 2012; Ye et al., 2008). Finally, the CDKN2B gene (p15INK4 gene) has been identified as a low-penetrance gene in MTC (Ruiz-Llorente et al., 2007). Thus these genetic analyses provide ample evidence that, in addition to RET/RAS somatic mutations, targeting of the Rb pathways through inactivation of CDK inhibitor family members contributes to human MTC tumorigenesis.

In our mouse model, NSE promoter-driven p25-GFP expression was predominantly detected in the thyroid. Only low levels of p25-GFP could be detected in lungs and adrenal gland and no primary tumors were observed in these tissues. The reason for this expression selectivity or possible sensitivity of C cells to Rb inactivation is presently unclear. However, we do not exclude a role for Cdk5 in other neuroendocrine cancers.

Neuroendocrine cancers are silent killers because they are difficult to diagnose due to a lack of symptoms and are often uncovered at advanced stages when window for effective surgical treatment has passed. Few treatment options are available due, in part, to incomplete understanding of the underlying molecular pathways and the lack of relevant animal models (Knostman et al., 2007). Existing models of MTC include transgenic mice bearing RET mutations (Cranston and Ponder, 2003) and animals deficient for Rb1/p53 (Harvey et al., 1995), prolactin receptor (Kedzia et al., 2005) or Rb1/Nras (Takahashi et al., 2006). However in most of these, constitutive transgene expression or gene knockout may introduce congenital confounds. In the model introduced here, MTC is reversibly and reproducibly induced in an adult with a fully developed and functional thyroid. Importantly, MTC originates from p25-mediated aberrant Cdk5 activation in C cells and not from a RET mutation. Hence, the animal model established here represents a clinically relevant model to study the onset and progression of sporadic MTC carcinogenesis. Furthermore, the ability to arrest the disease at various stages may facilitate the identification of druggable targets for therapy development. Finally this mouse model will be a useful preclinical tool for the development and testing of new adjuvant therapies for MTC (Dar et al., 2012; Wells et al., 2012).

EXPERIMENTAL PROCEDURES

Antibodies, siRNAs, plasmids and peptides

Antibodies for human calcitonin were from DAKO, GFP from Abcam, GADPH from Sigma, Cdk5, Cdk2, Cyclin A and p35/p25 from Santa Cruz Biotechnology. The Cdk5 monoclonal Ab was described by Lagace et al. (2008). The p35/p25 polyclonal antibody is directed to an antigen in the C-terminus of p35 and does not distinguish between p35 and p25. The specificity of p35/p25 antibody has been verified in brain tissues of p35 knockout animals (Figure S1B). Antibodies to total Rb, pRb-Ser807/811, STAT3 and pSTAT3 were from Cell Signaling Technology, and phospho-histone H1 from Millipore. Cdk5 siRNA was from Santa Cruz Biotechnology and p35 siRNA from Sigma. The kinase dead CDK5 construct, pCMV-KD-Cdk5 was previously described (Saito et al., 2007), pCMV-EGFP was from Clontech. The peptide was synthesized by the UT Southwestern Protein Chemistry Technology Center. The sequence of the Rb-Cdk5 small interfering peptide (SIP) was R7-PGGNIYISPLKSPYKISEGL and the control peptide R7-SYFHKEDRPPRDK.

Human Tissue Samples

Normal human and medullary thyroid specimens were obtained through a human subjects Institutional Review Board approved protocol UT Southwestern IRB 052004-044, “Molecular Analysis of Endocrine Tumors”. Written consent of subjects was obtained. Diagnosis of the neoplasm was confirmed by pathological review and RET-germline mutation analyses were obtained from MTC patient records. All MEN2A samples harbored germline point mutation in RET codon 634 resulting from a cysteine to tyrosine substitution.

Generation of NSE TetOp p25-GFP Mice

Bitransgenic mice were generated as described previously (Meyer et al., 2008). Briefly, the p25-GFP340 mouse strain, which contains a human p25-GFP transgene driven by the TetOp promoter (TetOp-p25-GFP), was crossed with the NSE5021 strain, which has a tetracycline transactivator (tTA) directed by the neural specific enolase promoter (NSE). This form of p25 is functional (Cruz et al., 2003) and the use of the tetOp system to drive NSE directed expression has been well characterized (Chen et al., 1998).

All mouse strains were maintained on a C57BL/6 background. Transgenic alleles were identified by a PCR-based genotyping strategy for p25-GFP and NSE-tTA alleles. Bitransgenic p25OE mice were positive for the NSE-tTA and p25-GFP transgenes. Control littermates were positive for NSE-tTA but not p25-GFP. Both groups were treated identically with regard to doxycycline administration. All subjects used in these studies were group-housed on a 12 hr light/dark cycle with access to food and water ad libitum. All procedures were performed during the light cycle, between 0600–1800 and were approved by the Institutional Animal Care and Use Committee of the University of Texas and conducted in accordance with the applicable portions of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Cell culture, transfections, drugs and SIP treatment

SHER-I, GRS-V, GRS-IV, RARE, SIN-J and MTC-SK cell lines were derived from human sporadic MTC tumors and maintained as described (Kaczirek et al., 2004; Pfragner et al., 1990; Pfragner et al., 2002). The MTCp25 mouse line was established by using MTC from mice that were overexpressing p25-GFP for at least 16 weeks. Normal human diploid fibroblasts were obtained from dissected mammary tissue using routine procedures. Cells were cultured in DMEM containing 10%FBS and were employed at passage 4. For drug treatments and small interfering peptide (SIP) assays, MTCp25 cells and each human cell lines were plated at a density of 2×105 cells/ml and incubated with different concentrations of doxycycline, CP681301, indolinone A, roscovitine and SIP for up to 5 days. The SIP contained a N-terminal poly-arginine (R7) Tag to penetrate the cell. Standard methods were used for transfections and soft agar assays. Detailed procedures can be found in the Supplemental Experimental Procedures.

Cell proliferation and viability assays

For proliferation analysis, cells were plated at a density of 2×105 cells/ml. The number of cells was determined on various days after plating by using a CASY®-1 Cell Counter & Analyzer TTC (Schärfe System). In the figures, ‘number of cells’ refers to the total number of cells that are counted in 1 ml cell suspension. Thus the ‘number of cells’ includes alive and dead cells. For cell viability assays, WST-1 Cell Proliferation Reagent (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) (Roche Diagnostics) was used as per the manufacturer’s instructions. Assays were performed in triplicate and repeated at least 2 times.

Immunoprecipitation-kinase assay

Proliferating mouse tumors were collected from mice deprived of doxycycline for at least 16 weeks. Arrested mouse tumors were obtained from mice that were off doxycycline for 16 weeks and replaced on doxycyline for 4 more weeks. MTCp25 cells were plated at a density of 2×105 cells/ml and treated, or not, with 10 μM doxycycline for 4 days. MTC-SK and SIN-J cells were plated at a density of 3×105 cells/ml and treated for 12 hr with CP681301 or transfected with pCMV-KD-Cdk5 or p35 siRNA. Transfected cells were harvested 24hr (pCMV-KD-Cdk5) or 5 days (p35 siRNA) post-transfection. Cdk5 or Cdk2 were immunoprecipitated from cell lysates by using anti-Cdk5 or anti-Cdk2 antibodies and their activity was assessed in vitro in saturating enzyme conditions and using histone H1 as a substrate. Detailed procedures can be found in the Supplemental Experimental Procedures.

Immunoblotting

Immunoblotting was carried out as previously described (Bibb et al., 1999).

Statistical Analysis

Data for individual assays represent the mean ± S.E.M. All experiments were designed with matched control conditions within each experiment to enable statistical comparison via two-tailed Student’s t-test and all value of p < 0.05 were considered statistically significant. GraphPad Prism 6.0 was used.

Supplementary Material

01

HIGHLIGHTS.

  • Cdk5 and its activators are expressed in human MTC

  • Cdk5 activity drives proliferation of sporadic human MTC cell lines

  • Transgenic expression of the Cdk5 cofactor, p25, rapidly induces lethal MTC in mice

  • Rb phosphorylation at Ser807/811 is critical for MTC progression

SIGNIFICANCE.

Neuroendocrine tumors are indolent malignancies arising from hormone-producing cells scattered throughout the body. MTC stems from the thyroid C cells and has a high mortality rate with rising incidence. Complete thyroid removal is the current primary therapy but recurrence is common and more effective treatments are needed. Here we show that Cdk5 promotes human MTC proliferation and that transgenic induction of aberrant Cdk5 activity in mouse thyroid C cells causes MTC. Furthermore, we reveal Rb protein as a downstream effector by which Cdk5 drives neuroendocrine cell neoplasia. Therefore, Cdk5 may be a promising drug target against MTC.

Acknowledgments

We thank G. Cote for MEN2A and control thyroid lysates, E. Knudsen for NDF cells and helpful advice, E. Nestler for NSE-Tta mice; K. Richter, L. Lau (Pfizer) for CP681301; F. Gillardon (Boehringer Ingelheim) for indolinone A; L. Meijer for roscovitine; and S. Hisanaga for the kinase-dead Cdk5 construct. We thank J. Shelton for histopathology advice, I. Mitchell, T. Singh T. Crone, and L. O’Connor for technical assistance and Antje Hillmann for reading the manuscript. This research was supported by a North American Neuroendocrine Tumor Society fellowship (KP), and U.S. National Institutes of Health Grants to L.H.T (NS051874), H.C. (CA121115 and CA109053), F.E.N. (GM067674), and J.A.B. (MH79710, MH083711, DA016672, DA033485, and NS073855); the Howard Hughes Medical Institute (L.H.T); and American Cancer Society MEN2 Thyroid Cancer Consortium Research Grants (RSGM-11-182-01, RPM-11-080-01 H.C.; RSGM-11-190-01, J.A.B.).

Footnotes

Authors contributions: KP, FP, JAR, JAB wrote the manuscript; K.P., E.C.R., F.P., R.P., J.A.B. designed experiments; experiments were performed and analyzed by K.P., C.T., J.G., L.N. for Figures 1, S1; by K.P., G.S., V.S. for Figures 2 and S2; E.C.R., D.M., E.R., J.A.G., P.A., J.A.R. for Figure 3; K.P., E.C.R., J.A.G, X.S., J.A.R. for Figures 4 and S3; K.P., E.C.R. for Figures 5 and S4; K.P., A.G. for Figures 6 and S5; G.M. maintained and genotyped the NSE/p25-GFP mouse line; L.H.T., S.S., B.G.R., H.C., F.E.N. and R.P. provided reagents.

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