Runx2 trans-activation mediated by the MSX2-interacting nuclear target requires homeodomain interacting protein kinase-3.

Runt-related transcription factor 2 (Runx2) and muscle segment homeobox homolog 2-interacting nuclear target (MINT) (Spen homolog) are transcriptional regulators critical for mammalian development. MINT enhances Runx2 activation of osteocalcin (OC) fibroblast growth factor (FGF) response element in an FGF2-dependent fashion in C3H10T1/2 cells. Although the MINT N-terminal RNA recognition motif domain contributes, the muscle segment homeobox homolog 2-interacting domain is sufficient for Runx2 activation. Intriguingly, Runx1 cannot replace Runx2 in this assay. To better understand this Runx2 signaling cascade, we performed structure-function analysis of the Runx2-MINT trans-activation relationship. Systematic truncation and domain swapping in Runx1:Runx2 chimeras identified that the unique Runx2 activation domain 3 (AD3), encompassed by residues 316-421, conveys MINT+FGF2 trans-activation in transfection assays. Ala mutagenesis of Runx2 Ser/Thr residues identified that S301 and T326 in AD3 are necessary for full MINT+FGF2 trans-activation. Conversely, phosphomimetic Asp substitution of these AD3 Ser/Thr residues enhanced activation by MINT. Adjacent Pro residues implicated regulation by a proline-directed protein kinase (PDPK). Systematic screening with PDPK inhibitors identified that the casein kinase-2/homeodomain-interacting protein kinase (HIPK)/dual specificity tyrosine phosphorylation regulated kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), but not ERK, c-Jun N-terminal kinase, p38MAPK, or other casein kinase-2 inhibitors, abrogated Runx2-, MINT-, and FGF2-activation. Systematic small interfering RNA-mediated silencing of DMAT-inhibited PDPKs revealed that HIPK3 depletion reduced MINT+FGF2-dependent activation of Runx2. HIPK3 and Runx2 coprecipitate after in vitro transcription-translation, and recombinant HIPK3 recognizes Runx2 AD3 as kinase substrate. Furthermore, DMAT treatment and HIPK3 RNAi inhibited MINT+FGF2 activation of Runx2 AD3, and nuclear HIPK3 colocalized with MINT. HIPK3 antisense oligodeoxynucleotide selectively reduced Runx2 protein accumulation and OC gene expression in C3H10T1/2 cells. Thus, HIPK3 participates in MINT+FGF2 regulation of Runx2 AD3 activity and controls Runx2-dependent OC expression.

Proline-directed protein kinase FA as a new signal transducing target for lethal cancer treatment.

Proline-directed protein kinase F(A) (PDPK F(A)) has been established as a multisubstrate/multifunctional PDPK essential for the development of highly malignant phenotypes and rapid progression of lethal cancers. The recent immunohistochemical, immunocytochemical and clinicopathologic studies combined demonstrate that overexpressed PDPK F(A) is dynamically and closely associated with the most aggressive malignant cells disseminating from primary tumors to peripheral blood, ascites, pleural effusions and second metastatic tumors of various types of cancer patients with poor prognosis. The antisense gene therapeutic studies further demonstrate that overexpressed PDPK F(A) is essential for the development of all aspects of neoplasia including highly metastatic spread, peritoneal dissemination, splenomegaly and chemoradioresistances. The inhibition of cancer progression together with the simultaneous enhancement of chemoradiosensitivities through the suppression of overexpressed PDPK F(A) by specific drug design may work synergistically with surgery and chemoradiotherapy to improve the poor survival rate and life quality of the patients with lethal malignancies. PDPK F(A), therefore, may represent the heel of Achilles and a new promising target for the strategic development of more efficacious treatment for lethal cancers

Proline-directed protein kinase FA as a potential target for diagnosis and therapy of human cancers.

Proline-directed protein kinase FA (PDPK FA) was originally identified as a phosphatase activating factor (FA) but has subsequently been characterized as a multisubstrate/multifunctional PDPK possibly associated with human cancers. In recent years, the immunohistochemical study revealed that PDPK FA was highly expressed in tumor mass and preferentially overexpressed in the invasive lesions of the resected tissue sections obtained from various types of cancer patients. The clinicopathologic study further revealed a close correlation of the overexpression of PDPK FA with poor prognosis of the cancer patients. The antisense gene therapy study also confirmed that due to its multisubstrate/multifunctional PDPK nature, the overexpression of PDPK FA is essential for the development of malignant growth, tumorigenesis, invasion, metastasis, anti-differentiation, anti-apoptosis and chemoresistance in human cancers. From immunohistochemical, clinicopathologic and antisense gene therapeutic studies combined together, PDPK FA has emerged as a key regulator of all aspects of neoplasia. In this way, nature provides prima facie evidence of a particular protein kinase's pivotal importance to the neoplastic state. PDPK FA therefore represents a newly-described, previously-undiscovered novel signal transducing target for diagnosis, disease monitoring, drug screening and therapy of human cancers.

Suppression of proline-directed protein kinase F(A) inhibits the malignant growth of human pancreatic ductal adenocarcinoma.

BACKGROUND: Proline-directed protein kinase F(A) (PDPK F(A)) was originally identified as a specific phosphatase activating factor, but has subsequently been demonstrated as a multisubstrate PDPK possibly involved in the regulation of diverse malignant characteristics of various types of human cancers including prostate, leukemia, bladder and colon cancers. However, the role of this PDPK in a lethal carcinoma, such as pancreatic ductal adenocarcinoma, remains to be established. MATERIALS AND METHODS: The stable antisense clones with specific suppression of overexpressed PDPK F(A) of human pancreatic ductal adenocarcinoma cells (MIA PaCa-2) were first selected and subsequently characterized for the in vitro and in vivo growth studies. RESULTS: The molecular and cellular studies revealed that the antisense clones of MIA PaCa-2 cells with specific suppression of overexpressed PDPK F(A) potentially exhibited cell growth retardation, decreased serum independence, poor clonogenic growth and loss of anchorage-independent growth. The in vivo study further confirmed that the SCID mice injected with the antisense clones with low-level PDPK F(A) did not develop any detectable tumors even after 7-week observation. In sharp contrast, the parental or control-transfected clones developed very large tumors (>5 cm3) under identical conditions. CONCLUSION: The molecular, cellular and animal results taken together demonstrate that overexpressed PDPK F(A) is essential for the malignant growth of human pancreatic ductal adenocarcinoma.