Maternal Embryonic Leucine Zipper Kinase (MELK) as a Novel Mediator and Biomarker of Radioresistance in Human Breast Cancer.

PURPOSE: While effective targeted therapies exist for estrogen receptor-positive and HER2-positive breast cancer, no such effective therapies exist for triple-negative breast cancer (TNBC); thus, it is clear that additional targets for radiosensitization and treatment are critically needed. EXPERIMENTAL DESIGN: Expression microarrays, qRT-PCR, and Western blotting were used to assess MELK RNA and protein expression levels. Clonogenic survival assays were used to quantitate the radiosensitivity of cell lines at baseline and after MELK inhibition. The effect of MELK knockdown on DNA damage repair kinetics was determined using γH2AX staining. The in vivo effect of MELK knockdown on radiosensitivity was performed using mouse xenograft models. Kaplan-Meier analysis was used to estimate local control and survival information, and a Cox proportional hazards model was constructed to identify potential factors impacting local recurrence-free survival. RESULTS: MELK expression is significantly elevated in breast cancer tissues compared with normal tissue as well as in TNBC compared with non-TNBC. MELK RNA and protein expression is significantly correlated with radioresistance in breast cancer cell lines. Inhibition of MELK (genetically and pharmacologically) induces radiation sensitivity in vitro and significantly delayed tumor growth in vivo in multiple models. Kaplan-Meier survival and multivariable analyses identify increasing MELK expression as being the strongest predictor of radioresistance and increased local recurrence in multiple independent datasets. CONCLUSIONS: Here, we identify MELK as a potential biomarker of radioresistance and target for radiosensitization in TNBC. Our results support the rationale for developing clinical strategies to inhibit MELK as a novel target in TNBC. Clin Cancer Res; 22(23); 5864-75. ©2016 AACR.

Direct regulation of osteocytic connexin 43 hemichannels through AKT kinase activated by mechanical stimulation.

Connexin (Cx) 43 hemichannels in osteocytes are thought to play a critical role in releasing bone modulators in response to mechanical loading, a process important for bone formation and remodeling. However, the underlying mechanism that regulates the opening of mechanosensitive hemichannels is largely unknown. We have recently shown that Cx43 and integrin α5 interact directly with each other, and activation of PI3K appears to be required for Cx43 hemichannel opening by mechanical stimulation. Here, we show that mechanical loading through fluid flow shear stress (FFSS) increased the level of active AKT, a downstream effector of PI3K, which is correlated with the opening of hemichannels. Both Cx43 and integrin α5 are directly phosphorylated by AKT. Inhibition of AKT activation significantly reduced FFSS-induced opening of hemichannels and disrupted the interaction between Cx43 and integrin α5. Moreover, AKT phosphorylation on Cx43 and integrin α5 enhanced their interaction. In contrast to the C terminus of wild-type Cx43, overexpression of the C-terminal mutant containing S373A, a consensus site previously shown to be phosphorylated by AKT, failed to bind with α5 and hence could not inhibit hemichannel opening. Together, our results suggest that AKT activated by FFSS directly phosphorylates Cx43 and integrin α5, and Ser-373 of Cx43 plays a predominant role in mediating the interaction between these two proteins and Cx43 hemichannel opening, a crucial step to mediate the anabolic function of mechanical loading in the bone.

14-3-3θ facilitates plasma membrane delivery and function of mechanosensitive connexin 43 hemichannels.

Intracellular signaling in osteocytes activated by mechanical loading is important for bone formation and remodeling. These signaling events are mediated by small modulators released from Cx43 hemichannels (HC). We have recently shown that integrin α5 senses the mechanical stimulation and induces the opening of Cx43 HC; however, the underlying mechanism is unknown. Here, we show that both Cx43 and integrin α5 interact with 14-3-3θ, and this interaction is required for the opening of Cx43 HC upon mechanical stress. The absence of 14-3-3θ prevented the interaction between Cx43 and integrin α5, and blocked HC opening. Furthermore, it decreased the transport of Cx43 and integrin α5 from the Golgi apparatus to the plasma membrane. Mechanical loading promoted the movement of Cx43 to the surface which was associated not only with an increase in 14-3-3θ levels but also its interaction with Cx43 and integrin α5. This stimulatory effect on forward transport by mechanical loading was attenuated in the absence of 14-3-3θ and the majority of the Cx43 accumulated in the Golgi. Disruption of the Golgi by brefeldin A reduced the association of Cx43 and integrin α5 with 14-3-3θ, further suggesting that the interaction is likely to occur in the Golgi. Together, these results define a previously unidentified, scaffolding role of 14-3-3θ in assisting the delivery of Cx43 and integrin α5 to the plasma membrane for the formation of mechanosensitive HC in osteocytes.

Biological role of connexin intercellular channels and hemichannels.

Gap junctions (GJ) and hemichannels (HC) formed from the protein subunits called connexins are transmembrane conduits for the exchange of small molecules and ions. Connexins and another group of HC-forming proteins, pannexins comprise the two families of transmembrane proteins ubiquitously distributed in vertebrates. Most cell types express more than one connexin or pannexin. While connexin expression and channel activity may vary as a function of physiological and pathological states of the cell and tissue, only a few studies suggest the involvement of pannexin HC in acquired pathological conditions. Importantly, genetic mutations in connexin appear to interfere with GJ and HC function which results in several diseases. Thus connexins could serve as potential drug target for therapeutic intervention. Growing evidence suggests that diseases resulting from HC dysfunction might open a new direction for development of specific HC reagents. This review provides a comprehensive overview of the current studies of GJ and HC formed by connexins and pannexins in various tissue and organ systems including heart, central nervous system, kidney, mammary glands, ovary, testis, lens, retina, inner ear, bone, cartilage, lung and liver. In addition, present knowledge of the role of GJ and HC in cell cycle progression, carcinogenesis and stem cell development is also discussed.

Mechanical stress-activated integrin α5ß1 induces opening of connexin 43 hemichannels.

The connexin 43 (Cx43) hemichannel (HC) in the mechanosensory osteocytes is a major portal for the release of factors responsible for the anabolic effects of mechanical loading on bone formation and remodeling. However, little is known about how the Cx43 molecule responds to mechanical stimulation leading to the opening of the HC. Here, we demonstrate that integrin α5ß1 interacts directly with Cx43 and that this interaction is required for mechanical stimulation-induced opening of the Cx43 HC. Direct mechanical perturbation via magnetic beads or conformational activation of integrin α5ß1 leads to the opening of the Cx43 HC, and this role of the integrin is independent of its association with an extracellular fibronectin substrate. PI3K signaling is responsible for the shear stress-induced conformational activation of integrin α5ß1 leading to the opening of the HC. These results identify an unconventional function of integrin that acts as a mechanical tether to induce opening of the HC and provide a mechanism connecting the effect of mechanical forces directly to anabolic function of the bone.

Gap junctions and hemichannels in signal transmission, function and development of bone.

Gap junctional intercellular communication (GJIC) mediated by connexins, in particular connexin 43 (Cx43), plays important roles in regulating signal transmission among different bone cells and thereby regulates development, differentiation, modeling and remodeling of the bone. GJIC regulates osteoblast formation, differentiation, survival and apoptosis. Osteoclast formation and resorptive ability are also reported to be modulated by GJIC. Furthermore, osteocytes utilize GJIC to coordinate bone remodeling in response to anabolic factors and mechanical loading. Apart from gap junctions, connexins also form hemichannels, which are localized on the cell surface and function independently of the gap junction channels. Both these channels mediate the transfer of molecules smaller than 1.2kDa including small ions, metabolites, ATP, prostaglandin and IP(3). The biological importance of the communication mediated by connexin-forming channels in bone development is revealed by the low bone mass and osteoblast dysfunction in the Cx43-null mice and the skeletal malformations observed in occulodentodigital dysplasia (ODDD) caused by mutations in the Cx43 gene. The current review summarizes the role of gap junctions and hemichannels in regulating signaling, function and development of bone cells. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

"INTEGRINating" the connexin hemichannel function in bone osteocytes through the action of integrin α5.

Mechanical loading influences skeletal structural integrity and bone remodeling. Application of a mechanical stimulus such as fluid flow shear stress to the bone osteocytes activates the cascade of mechanotransduction mediated by multiple signaling molecules. Hemichannels formed by connexin molecules are emerging as a candidate mechanosensor. Connexin 43 (Cx43) hemichannels open in response to mechanical stimulation to release bone modulators which influence bone remodeling. Our study identified a direct interaction between integrin α5 and Cx43 which was essential for hemichannels to open. Uncoupling the interaction blocked the hemichannels and shear stress enhanced the interaction between the two proteins to promote channel opening. More importantly, integrin α5, independent of its association with fibronectin, was activated upon shear stress through a PI3K signaling pathway. These results suggest a critical regulatory mechanism for Cx43 hemichannel opening through the association of integrin α5, resulting in release of bone anabolic factors required for bone development.

Prostaglandin promotion of osteocyte gap junction function through transcriptional regulation of connexin 43 by glycogen synthase kinase 3/beta-catenin signaling.

Gap junction intercellular communication in osteocytes plays an important role in bone remodeling in response to mechanical loading; however, the responsible molecular mechanisms remain largely unknown. Here, we show that phosphoinositide-3 kinase (PI3K)/Akt signaling activated by fluid flow shear stress and prostaglandin E(2) (PGE(2)) had a stimulatory effect on both connexin 43 (Cx43) mRNA and protein expression. PGE(2) inactivated glycogen synthase kinase 3 (GSK-3) and promoted nuclear localization and accumulation of beta-catenin. Knockdown of beta-catenin expression resulted in a reduction in Cx43 protein. Furthermore, the chromatin immunoprecipitation (ChIP) assay demonstrated an association of beta-catenin with the Cx43 promoter, suggesting that beta-catenin could regulate Cx43 expression at the level of gene transcription. We have previously reported that PGE(2) activates cyclic AMP (cAMP)-protein kinase A (PKA) signaling and increases Cx43 and gap junctions. Interestingly, the activation of PI3K/Akt appeared to be independent of the activation of PKA, whereas both PI3K/Akt and PKA signaling inactivated GSK-3 and increased beta-catenin translocation. Together, these results suggest that shear stress, through PGE(2) release, activates both PI3K/Akt and cAMP-PKA signaling, which converge through the inactivation of GSK-3, leading to the increase in nuclear accumulation of beta-catenin. beta-Catenin binds to the Cx43 promoter, stimulating Cx43 expression and functional gap junctions between osteocytes.