clinically-relevant-transplantable-tumor-models-for-studying

Clinically Relevant Transplantable Tumor Models for Studying Metastatic Ovarian Cancer and Therapeutic Response in Mouse

Metastatic ovarian cancer is an extremely lethal gynecologic malignancy that lacks effective treatment options. Development of clinically translatable ...

University of Maryland Baltimore County

University of Maryland Greenebaum Comprehensive Cancer Center

Elucidating kinase-substrate relationships is critical for understanding how phosphorylation affects signal transduction and regulatory cascades. Using the alpha catalytic subunit of protein kinase CK2 (CK2alpha) as a paradigm, we developed an in-gel method to facilitate identification of physiologic kinase substrates. In this approach, the roles of kinase and substrate in a classic in-gel kinase assay are reversed. In the reverse in-gel kinase assay (RIKA), a kinase is copolymerized in a denaturing polyacrylamide gel used to resolve a tissue or cell protein extract. Restoration of kinase activity and substrate structure followed by an in situ kinase reaction and mass spectrometric analyses results in identification of potential kinase substrates. We demonstrate that this method can be used to profile both known and novel human and mouse substrates of CK2alpha and cAMP-dependent protein kinase (PKA). Using widely available straightforward technology, the RIKA has the potential to facilitate discovery of physiologic kinase substrates in any biological system.

The reverse in-gel kinase assay to profile physiological kinase substrates.

Loss of NKX3.1 is an early and consistent event in prostate cancer and is associated with increased proliferation of prostate epithelial cells and poor prognosis. NKX3.1 stability is regulated post-translationally through phosphorylation at multiple sites by several protein kinases. Here, we report the paradoxical stabilization of the prostate-specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim-1 in prostate cancer cells. Pharmacologic Pim-1 inhibition using the small molecule inhibitor CX-6258 decreased steady state levels and half-life of NKX3.1 protein but mRNA was not affected. This effect was reversed by inhibition of the 26S-proteasome, demonstrating that Pim-1 protects NKX3.1 from proteasome-mediated degradation. Mass spectrometric analyses revealed Thr89, Ser185, Ser186, Ser195, and Ser196 as Pim-1 phospho-acceptor sites on NKX3.1. Through mutational analysis, we determined that NKX3.1 phosphorylation at Ser185, Ser186, and within the N-terminal PEST domain is essential for Pim-1-mediated stabilization. Further, we also identified Lys182 as a critical residue for NKX3.1 stabilization by Pim-1. Pim-1-mediated NKX3.1 stabilization may be important in maintaining normal cellular homeostasis in normal prostate epithelial cells, and may maintain basal NKX3.1 protein levels in prostate cancer cells.

Stabilization of the prostate-specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim-1 in prostate cancer cells.

MYC levels are tightly regulated in cells, and deregulation is associated with many cancers. In this report, we describe the existence of a MYC-protein kinase A (PKA)-polo-like kinase 1 (PLK1) signaling loop in cells. We report that sequential MYC phosphorylation by PKA and PLK1 protects MYC from proteasome-mediated degradation. Interestingly, short term pan-PKA inhibition diminishes MYC level, whereas prolonged PKA catalytic subunit α (PKACα) knockdown, but not PKA catalytic subunit β (PKACβ) knockdown, increases MYC. We show that the short term effect of pan-PKA inhibition on MYC is post-translational and the PKACα-specific long term effect on MYC is transcriptional. These data also reveal distinct functional roles among PKA catalytic isoforms in MYC regulation. We attribute this effect to differential phosphorylation selectivity among PKA catalytic subunits, which we demonstrate for multiple substrates. Further, we also show that MYC up-regulates PKACβ, transcriptionally forming a proximate positive feedback loop. These results establish PKA as a regulator of MYC and highlight the distinct biological roles of the different PKA catalytic subunits.

Protein kinase A regulates MYC protein through transcriptional and post-translational mechanisms in a catalytic subunit isoform-specific manner.

The androgen-regulated homeodomain transcription factor NKX3.1 plays roles in early prostate development and functions as a prostate-specific tumor suppressor. Decreased expression of NKX3.1 protein is common in primary prostate cancer. Discordance between NKX3.1 mRNA and protein levels during prostate carcinogenesis suggested a key role for post-transcriptional modifications in regulating NKX3.1 protein levels in prostate epithelial cells. Subsequent studies revealed NKX3.1 to be modified post-translationally at multiple sites.

Regulating NKX3.1 stability and function: Post-translational modifications and structural determinants.

Biology of ovarian cancer: Models, microenvironment, and therapeutics: 15th Biennial Rivkin Center Ovarian Cancer Research Symposium.

Ovarian cancer is characterized by aggressive metastasis, chemoresistance, and poor survival outcomes. Gaps in understanding of factors that drive these phenotypes have hindered the development of actionable therapeutic targets. We demonstrate that Zinc Finger Protein 217 (ZNF217) is a key pro-metastatic factor in ovarian cancer cells. ZNF217 overexpression dramatically increases proliferation, metastasis, and chemoresistance while its depletion impairs these phenotypes. Consistently, ZNF217 overexpression is associated with poor prognosis in both mouse models and ovarian cancer patients. Interestingly, ZNF217 induces metastatic phenotypes in fallopian tube cells, suggesting potential role in the transition of early-stage tumors to aggressive carcinoma. ZNF217's oncogenic activity is dependent on its ability to bind DNA and alter multiple processes, including EMT, that are critical in driving different aspects of cancer progression. Thus, our data establishes ZNF217 as a potent oncogene in ovarian cancer cells that impacts multiple steps in the metastatic process and a potential therapeutic target.

Article	Year
The reverse in-gel kinase assay to profile physiological kinase substrates. Nature methods\| PubMed ID: 17952089	2007
Stabilization of the prostate-specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim-1 in prostate cancer cells. Journal of cellular biochemistry\| PubMed ID: 23129228	2013
Protein kinase A regulates MYC protein through transcriptional and post-translational mechanisms in a catalytic subunit isoform-specific manner. The Journal of biological chemistry\| PubMed ID: 23504319	2013
Regulating NKX3.1 stability and function: Post-translational modifications and structural determinants. The Prostate\| PubMed ID: 26841725	2016
Biology of ovarian cancer: Models, microenvironment, and therapeutics: 15th Biennial Rivkin Center Ovarian Cancer Research Symposium. Gynecologic oncology\| PubMed ID: 40896859	2025
ZNF217 promotes ovarian cancer progression by impacting multiple pivotal steps in the metastatic process. NPJ precision oncology\| PubMed ID: 41345234	2025

Achuth Padmanabhan

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