p12^CDK2-AP1 in embryo development

Despite of accumulating evidences that p12 functions as an inhibitory molecule in cell cycle regulation, the normal physiological role of p12 has not been clearly established. It has not been addressed why the cells need a significant level of growth inhibitory p12 throughout the lifespan. Studies with p12-/- murine ES cells showed that a targeted disruption of p12 resulted in an increased cellular proliferation along with an enhanced CDK2 activity and remarkable hyperphosphorylation of pRB. Also, we have been able to generate p12+/- mice, but failed to establish p12-/- progeny due to an early embryonic lethality. In addition, expression microarray analysis on p12^-/- cells revealed significant alterations in the expression of genes involved in embryogenesis and spermatogenesis. It is hypothesized that a proper temporal expression of p12 plays an essential role in the early embryogenesis and in addition a disturbed regulation of p12 in fully differentiated cells could lead to the transformation of cells. However, the underlying mechanism needs to be defined. Based on our previous studies, molecular interaction with CDK2 is a pivotal event in p12 biology and a disrupted interaction between CDK2 and p12 may trigger a cascade of altered signaling leading to altered phenotype including embryonic lethality. Downstream molecular event of p12-CDK2 is pRB regulation. Currently, it is known that knockout of CDK2 is viable, but knockout of RB is embryonic lethal. Inactivation of Rb in mice results in unscheduled cell proliferation, apoptosis and widespread developmental defects, leading to embryonic death by day 14.5.  Recent study showed that loss of RB leads to excessive proliferation of trophoblast cells and a severe disruption of the normal labyrinth architecture in the placenta. This is accompanied by a decrease in vascularization and a reduction in placental transport function. As evidenced in ES p12-/- cells showing hyperphosphorylation of pRB, the knockout of p12 may lead to a constitutive inactivation of RB and could potentially mimic the situation of loss of RB during embryo development. This would be addressed by using conditional knockout strategy and also rescue experiment during early onset of gestation period.

Translational application of p12^CDK2-AP1

 p12 gene therapy significantly induced antitumor effects as compared with controls, including (a) size and weight of p12-treated tumors decreased by 51% to 72% compared with all controls (P < 0.02), (b) tumor growth rate post-therapy was inhibited by 55% to 64% compared with empty vector controls (P < 0.0001), and (c) p12 expression was higher in p12-treated than controls (P < 0.002) by two-tailed t test analyses. Mechanistically, p12 treatment affected cell turnover kinetics as assessed by apoptotic and cell proliferation indices. p12 therapy significantly increased terminal nucleotidyl transferas mediated nick end labeling (P < 0.05) and morphology-based apoptotic indices (P < 0.05) as well as significantly decreased Ki-67 cell proliferation indices (P < 0.001) compared with controls, resulting in a net cell turnover reduction in p12-treated tumors. We show that this novel therapeutic modality can significantly induce antitumor responses in vivo. These results support a role for p12 as a novel tumor growth suppressor gene therapy and suggest that optimization and/or combination with current therapies may hold considerable promise in preparation for clinical trials.

Cyclin G2

Using expression microarray we have previously shown that human cyclin G2 (hCG2) is significantly downregulated in laser capture microdissected (LCM) oral cancer epithelia (Alevizos et al., Oncogene 20: 6196-6204, 2001). Western analysis showed detectable hCG2 protein in normal (2/2) but not in malignant (4/4) oral keratinocyte cell lines. Immunohistochemistry analysis performed on oral cancers showed that normal oral mucosa (100%, 12/12) and 69.1% (47/68) of dysplastic oral epithelia expressed readily detectable hCG2 in the nuclei. However, only 11.1% of oral cancer epithelia (14/126) showed mild hCG2 nuclear staining. Interestingly, of the oral cancers devoid of nuclear hCG2 (112 cases), 58 cases (52%) showed cytoplasmic hCG2 immunostaining whereas the other 54 cases (48%) exhibited neither nuclear nor cytoplasmic hCG2 staining. In vitro functional study by ectopic restoration of hCG2 expression in the human malignant squamous cell carcinoma line SCC15 resulted in a significant inhibition of cellular proliferation (p<0.001) and colony formation (p<2x10^-5) with increased population of G1 phase and decreased in S phase (p<0.01). Furthermore, stable downregulation of hCG2 by short interference RNA (siRNA) based gene silencing in immortalized normal oral keratinocytes resulted in enhanced cell growth with increase in S and prominently in G2 phase. Since hCG2 has been implicated as a negative regulator in cell cycle progression, our results support that hCG2 dysregulation may play an important role in epithelial transformation and the early stages of human oral cancer development. We are currently working on the identification of potential hCG2 interacting partners to better understand the mechanism of hCG2 in cell cycle regulation.

Funding

RO1-DE14857-02 07/01/2003-04/30/2007
NIH/NIDCR/NCI
p12CDK2-AP1 in Cell Cycle Control and Oral Carcinogenesis
The aims of this proposal include characterizing the in vivo role of p12CDK2-AP1 as a negative regulator of CDK2 activities and determining how this interaction is involved in the TGF- antiproliferative pathway.

Publications

1. Todd R, McBride J, Tsuji T, Donoff RB, Nagai M, Chou MY, Chiang T, and Wong DTW.  Deleted in oral cancer-1 (doc-1), a candidate oral cancer suppressor gene (1995).  FASEB J 9, 1362-70.

2. Tsuji T, Duh F-M, Latif FL, Popescu NC, Zimonjic DB, McBride J, Rheinwald JG, Matsuo KM, Ohyama HO, Todd RT, Nagata EN, Terakado N, Sasaki A, Matsumura T, Lerman MI, Wong DTW. Cloning, Mapping, Expression, Function and Mutation Analyses of the Human Ortholog of the Hamster Putative Tumor Suppressor Gene doc-1 (1998). J Biol Chem 273: 6704-6709.

3. Matsuo K, Shintani S, Tsuji T, Nagata E, Lerman M, McBride J, Nakahara Y, Todd R, Wong DTW. p12^DOC-1, a growth suppressor, associates with DNA polymerase /primase (2000). FASEB J. 14: 1318-1324.

4. Shintani S, Ohyama H, Zhang X, McBride J, Matsuo K, Tsuji T, Todd R, Lerman M, Wong DTW. p12^DOC-1 is a novel CDK2-associated protein (2000). Mol Cell Biol  20:6300-6307.

5. Shintani S, Mihara M, Terakado N, Nakahara Y, Matsumura T, Kohno Y, Ohyama H, McBride J, Kent R, Todd R, Tsuji T, Wong DTW. Reduction of p12^DOC-1 expression is a negative prognostic indicator in Japanese patients with surgically resected oral cancer (2001). Clin Cancer Res 7: 2776-2782.

6. Kohno, Y., Patel, V., Kim, Y., Tsuji, T., Chin, B.-R., Sun, M., Donoff, R.B., Kent, R., Wong, D.T.W., and Todd, R. Apotosis, proliferation and p12^doc-1 profiles in normal, dysplastic and malignant squamous epithelium of the Syrian hamster cheek pouch model (2002). Oral Oncol 38(3):274-280.

7. Hu, M.G., Hu, G.-F., Kim, Y., Takanori, T., McBride, J., Hinds, P., and Wong. D.T.W. Role of p12^CDK2-AP1 in TGF-b1-mediated Growth Suppression (2004). Cancer Res 64(2):490-499.

8. Buajeeb, W., Zhang, X., Ohyama, H., Han, D., Surarit, R., Kim, Y., Wong, D.T.W. Interaction of the CDK2 associated protein-1, p12DOC-1/CDK2AP1, with its homolog, p14DOC-1R (2004). Biochem Biophys Res Commun 315(4):998-1003.

9. Kim, Y., Shintani, S., Kohno, Y., Zhang, R., and Wong, D.T. Cyclin G2 dysregulation in human oral cancer (2004). Cancer Res 64(24): 8980-8986.

10. Kim, Y., Ohyama, H., Patel, V., Figueiredo, M.L., and Wong, D.T. Mutation of Cys¹⁰⁵ inhibits dimerization of   p12^CDK2-AP1 and its growth suppressor effect (2005). J Biol Chem 280(24):23273-79.