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Background-The canonical PI3K/Akt pathway is hyperactive in a variety of cancers including ovarian cancer (OVCa). Using baculovirus-expressed, FPLC-purified enzymes, we previously demonstrated that Calcium/Calmodulin-dependent CaM Kinase Kinase 2 (CaMKK2) activates the oncogene Akt by direct phosphorylation of its primary activation site T308. In OVCAR-3 and other OVCa cells, RNAi-mediated knockdown of CaMKK2 decreased basal Akt phosphorylation at T308 in a Phosphoinositide-Dependent Kinase-1 (PDK1)-independent manner. CaMKK2 knockdown also decreased Akt phosphorylation at its secondary activation site, S473 by an apparently indirect mechanism. Results- In this study we show that CaMKK2 mediates downstream effects of epidermal growth factor (EGF) on the Akt cascade in OVCa cells. Treatment of OVCAR3 cells with EGF produced a prominent enhancement of p-Akt T308 which was reduced ~2-fold, by either CaMKK2 or PDK1 knockdowns. Knockdown of CaMKK2 but not PDK1 reduced p-Akt S473 after EGF treatment. Similar results were obtained with Insulin-like growth factor 1 (IGF1). To discern how CaMKK2 is engaged by EGF action we examined whether, in vitro, CaMKK2 phosphorylation of WT Akt or a synthetic peptide encompassing T308 is stimulated by Calcium/CaM and/or Phosphatidylinositol (3,4,5)-trisphosphate (PIP3). CaMKK2 activity was enhanced by Calcium/CaM but not by soluble PIP3. CaMKK2 did not bind to a dot blot of various phosphoinositides, although strong binding to PIP3 was observed with PDK1. Alternatively, Akt regulation by CaMKK2 may occur via a Calcium/CaM-dependent pathway. Increased p-Akt T308 and S473 upon treatment of cells with the calcium-ionophore, ionomycin, was significantly reduced by CaMKK2 knockdown. EGF stimulation of Akt phosphorylation at both T308 and S473 was significantly reduced by the intracellular calcium chelator, BAPTA-AM and by the CaM inhibitor, W7. We also probed the mechanism by which optimal cellular Akt phosphorylation at S473 requires CaMKK2. mTOR complex 2 (mTORC2) and DNA-dependent protein kinase (DNA-PKcs) are considered candidate kinases for p-Akt S473. CaMKK2 knockdown regulated p-Akt S473 in M059J cells, a DNA-PKcs null cell line similarly to that observed with OVCAR3 cells, suggesting that mTORC2 may be regulated by CaMKK2. Using an MS/MS Protein-protein interaction (PPI) screen we observed CaMMK2 interaction with multiple ribosomal proteins an observation consistent with suggested mTORC2 ribosomal regulation of Akt S473 phosphorylation. Conclusions-This study documents Akt activating phosphorylation by CaMKK2 in response to EGF in a Calcium/CaM-dependent, PI3K/PIP3 independent, manner. Besides directly phosphorylating Akt at T308, CaMKK2 regulates p-Akt S473 potentially through the regulation of mTORC2 activity. Our results highlight the importance of CaMKK2 activated by growth factor signals as a parallel system to the oncogenic PI3K/PDK1 pathway. Citation Format: Shuhang Dai, Angela M. Gocher, Arthur M. Edelman. CaMKK2 regulates EGF-dependent activation of oncogenic Akt in ovarian cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1430.

Knee osteoarthritis (KOA) is a degenerative joint disorder manifested with deformity, pain, and functional disability due to damage of the articular cartilage. Cell therapy with mesenchymal stem cells (MSCs) holds great promise to alleviate or even cure the degenerative diseases including KOA. However, the evidence of efficacy of human adipose tissue-derived MSCs (hAdMSCs) on KOA therapy remains limited. Here, we evaluate the therapeutic efficacy of hAdMSCs for KOA, using a medial meniscal transection (MMT) rat model. Our study demonstrated that intra-articular injection of 1.25×10(6) hAdMSCs significantly attenuated MMT-induced joint pain in a KOA rats model. The results of this study provide strong evidence that hAdMSCs-based therapy can be regarded as a prominent treatment option for patients with KOA.

Hyperactivation of Akt is associated with oncogenic changes in the growth, survival, and chemoresistance of cancer cells. The PI3K/phosphoinositide-dependent kinase (PDK) 1 pathway represents the canonical mechanism for phosphorylation of Akt at its primary activation site, Thr-308. We observed that Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (β) (CaMKK2) is highly expressed in high-grade serous ovarian cancer, and we investigated its role in Akt activation in ovarian cancer (OVCa) cell lines (OVCAR-3, SKOV-3, and Caov-3). Knockdown or pharmacological inhibition of CaMKK2 produced phenotypes expected of Akt inhibition, including reductions in cell growth and cell viability and in the regulation of Akt downstream targets involved in G1/S transition and apoptosis. CaMKK2 knockdown or inhibition decreased Akt phosphorylation at Thr-308 and Ser-473 to extents similar to those of PDK1 knockdown or PI3K inhibition. Combined CaMKK2 and PDK1 knockdown or CaMKK and PI3K inhibition, respectively, produced additive effects on p-Akt and cell growth, consistent with direct Akt phosphorylation by CaMKK2. This conclusion was supported by the absence of effects of CaMKK2 knockdown/inhibition on alternative means of activating Akt via p-Akt Thr-450, p-PDK1 Ser-241, or p-IRS1 Ser-636/639. Recombinant CaMKK2 directly activated recombinant Akt by phosphorylation at Thr-308 in a Ca2+/CaM-dependent manner. In OVCa cells, p-Akt Thr-308 was significantly inhibited by intracellular Ca2+i chelation or CaM inhibition. Ionomycin-induced Ca2+ influx promoted p-Akt, an effect blocked by PDK1, and/or CaMKK2, siRNAs, and by PI3K and/or CaMKK inhibitors. CaMKK2 knockdown potentiated the effects of the chemotherapeutic drugs carboplatin and PX-866 to reduce proliferation and survival of OVCa cells.

Uniform gold nanostars (Au NS) were conjugated with cyclic RGD (cRGD) and near infrared (NIR) fluorescence probe (MPA) or anti-cancer drug (DOX) to obtain multi-functional nanoconstructs, Au-cRGD-MPA and Au-cRGD-DOX respectively. The NIR contrast agent Au-cRGD-MPA was shown to have low cytotoxicity. Using tumor cells and tumor bearing mice, these imaging nanoparticles demonstrated favorable tumor-targeting capability mediated by RGD peptide binding to its over-expressed receptor on the tumor cells. The multi-therapeutic analogue, Au-cRGD-DOX, integrates targeting tumor, chemotherapy and photo-thermotherapy into a single system. The synergistic effect of photo-thermal therapy and chemotherapy was demonstrated in different tumor cell lines and in vivo using S180 tumor-bearing mouse models. The viability of MDA-MB-231 cells was only 40 % after incubation with Au-cRGD-DOX and irradiation with NIR light. Both tail vein and intratumoral injections showed Au-cRGD-DOX treated mice exhibiting the slowest tumor increase. These results indicate that the multifunctional nanoconstruct is a promising combined therapeutic agent for tumor-targeting treatment, with the potential to enhance the anti-cancer treatment outcomes.

Re-activation of the transcriptional activity of the androgen receptor (AR) is an important factor mediating progression from androgen-responsive to castrate-resistant prostate cancer (CRPC). However, the mechanisms regulating AR activity in CRPC remain incompletely understood. Ca(2+) /calmodulin-dependent kinase kinase (CaMKK) 2 was previously shown to regulate AR activity in androgen-responsive prostate cancer cells. Our objective was to further explore the basis of this regulation in CRPC cells.The abundance of CaMKK2 in nuclear fractions of androgen-responsive prostate cancer and CRPC, cells were determined by subcellular fractionation and Western blotting. CaMKK2 association with nuclear pore complexes (NPCs) and nucleoporins (Nups) including Nup62, were imaged by structured illumination and super-resolution fluorescence microscopy and co-immunoprecipitation, respectively. The abundance and subcellular localization of CaMKK2 and Nup62 in human clinical specimens of prostate cancer was visualized by immunohistochemistry. The role of Nups in the growth and viability of CRPC cells was assessed by RNA interference and cell counting. The involvement of CaMKK2 and Nup62 in regulating AR transcriptional activity was addressed by RNA interference, chromatin immunoprecipitation, androgen response element reporter assay, and Western blotting.CaMKK2 was expressed at higher levels in the nuclear fraction of CPRC C4-2 cells, than in that of androgen-responsive LNCaP cells. In C4-2 cells, CaMKK2 associated with NPCs of the nuclear envelope and physically interacted with Nup62. CaMKK2 and Nup62 demonstrated pronounced, and similar increases in both expression and perinuclear/nuclear localization in human clinical specimens of advanced prostate cancer relative to normal prostate. Knockdown of Nup62, but not of Nups, 98 or 88, reduced growth and viability of C4-2 cells. Knockdown of Nup62 produced a greater reduction of the growth and viability of C4-2 cells than of non-neoplastic RWPE-1 prostatic cells. Nup62, CaMKK2, and the AR were recruited to androgen response elements of the AR target genes, prostate specific antigen, and transmembrane protease, serine 2. Knockdown of CaMKK2 and Nup62 reduced prostate specific antigen expression and AR transcriptional activity driven by androgen response elements from the prostate-specific probasin gene promoter.Nup62 and CaMKK2 are required for optimal AR transcriptional activity and a potential mechanism for AR re-activation in CRPC.

Two kinds of thermally responsive polymers P(FAA-NIPA-co-AAm-co-ODA) and P(FPA-NIPA-co-AAm-co-ODA) containing folate, isopropyl acrylamide and octadecyl acrylate were fabricated through free radical random copolymerization for targeted drug delivery. Then the micelles formed in aqueous solution by self-assembly and were characterized in terms of particle size, lower critical solution temperature (LCST) and a variety of optical spectra. MTT assays demonstrated the low cytotoxicity of the control micelle and drug-loaded micelle on A549 cells and Bel 7402 cells. Then fluorescein and cypate were used as model drugs to optimize the constituents of micelles for drug entrapment efficiency and investigate the release kinetics of micelles in vitro. The FA and thermal co-mediated tumor-targeting efficiency of the two kinds of micelles were verified and compared in detail at cell level and animal level, respectively. These results indicated that the dual-targeting micelles are promising drug delivery systems for tumor-targeting therapy.