Calcium/calmodulin-dependent protein kinase kinase 2 mediates pleiotropic effects of epidermal growth factor in cancer cells.

AIMS: Engagement of epidermal growth factor (EGF) with its receptor (EGFR) produces a broad range of cancer phenotypes. The overriding aim of this study was to understand EGFR signaling and its regulation by the Ca2+/calmodulin (CaM) dependent protein kinase kinase 2 (CaMKK2) in cancer cells. RESULTS: In ovarian cancer cells and other cancer cell types, EGF-induced activation of oncogenic Akt is mediated by both the canonical PI3K-PDK1 pathway and by CaMKK2. Akt activation induced by EGF occurs by both calcium-dependent and calcium-independent mechanisms. In contrast to the canonical pathway, CaMKK2 neither binds to, nor is regulated by phosphoinositides but is activated by Ca2+/CaM. Akt activation at its primary activation site, T308 occurs by direct phosphorylation by CaMKK2, but activation at its secondary site (S473), is through an indirect mechanism requiring mTORC2. In cells in which another CaMKK2 target, 5'AMP-dependent protein kinase (AMPK) was deleted, Akt activation and calcium-dependency of activation were still observed. CaMKK2 accumulates in the nucleus in response to EGF and regulates transcription of phosphofructokinase platelet (PFKP) a glycolytic regulator. CaMKK2 is required for optimal PFK activity. CaMKK2 regulates transcription of plasminogen activator, urokinase (PLAU) a metastasis regulator. The EGFR inhibitor gefitinib synergizes with CaMKK2 inhibition in the regulation of cell survival and increases the dose-reduction index. CRISPR/Cas9 knockout of CaMKK2 leads to compensatory PTEN downregulation and upregulation of Akt activation. CONCLUSIONS: CaMKK2-mediation of EGFR action may enable cancer cells to use intracellular calcium elevation as a signal for growth and survival.

Comparative Analysis of the Therapeutic Effects of Amniotic Membrane and Umbilical Cord Derived Mesenchymal Stem Cells for the Treatment of Type 2 Diabetes.

Type 2 diabetes mellitus (T2DM), one of the most common carbohydrate metabolism disorders, is characterized by chronic hyperglycemia and insulin resistance (IR), and has become an urgent global health challenge. Mesenchymal stem cells (MSCs) originating from perinatal tissues such as umbilical cord (UC) and amniotic membrane (AM) serve as ideal candidates for the treatment of T2DM due to their great advantages in terms of abundant source, proliferation capacity, immunomodulation and plasticity for insulin-producing cell differentiation. However, the optimally perinatal MSC source to treat T2DM remains elusive. This study aims to compare the therapeutic efficacy of MSCs derived from AM and UC (AMMSCs and UCMSCs) of the same donor in the alleviation of T2DM symptoms and explore the underlying mechanisms. Our results showed that AMMSCs and UCMSCs displayed indistinguishable immunophenotype and multi-lineage differentiation potential, but UCMSCs had a much higher expansion capacity than AMMSCs. Moreover, we uncovered that single-dose intravenous injection of either AMMSCs or UCMSCs could comparably reduce hyperglycemia and improve IR in T2DM db/db mice. Mechanistic investigations revealed that either AMMSC or UCMSC infusion could greatly improve glycolipid metabolism in the liver of db/db mice, which was evidenced by decreased liver to body weight ratio, reduced lipid accumulation, upregulated glycogen synthesis, and increased Akt phosphorylation. Taken together, these data indicate that the same donor-derived AMMSCs and UCMSCs possessed comparable effects and shared a similar hepatoprotective mechanism on the alleviation of T2DM symptoms.

Distinguishing parallel from series circuits with a double knockdown procedure in human cell lines.

Signaling cascades can act in series or in parallel. Here, we describe a convenient and robust protocol for dual, sequential knockdown of two proteins using RNA interference. We detail the steps for a quantitative mapping of signaling circuitry. We used this approach to study kinases in human ovarian cancer cells, but the protocol can be applied to many other posttranslational modifications. For complete details on the use and execution of this protocol, please refer to Gocher et al. (2017).1.

Alleviation of medial meniscal transection-induced osteoarthritis pain in rats by human adipose derived mesenchymal stem cells.

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 1.25×106 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.

Akt activation by Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) in ovarian cancer cells.

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 (ß) (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.

Nucleoporin 62 and Ca(2+)/calmodulin dependent kinase kinase 2 regulate androgen receptor activity in castrate resistant prostate cancer cells.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Nup62 and CaMKK2 are required for optimal AR transcriptional activity and a potential mechanism for AR re-activation in CRPC.

Thermal responsive micelles for dual tumor-targeting imaging and therapy.

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.

Multifunctional gold nanostar conjugates for tumor imaging and combined photothermal and chemo-therapy.

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.