A Potential Role for Integrin-Linked Kinase in Colorectal Cancer Growth and Progression via Regulating Senescence and Immunity

Integrin-linked kinase (ILK) has been implicated as a molecular driver and mediator in both inflammation and tumorigenesis of the colon. ILK functions as an adaptor and mediator protein linking the extracellular matrix with downstream signaling pathways. ILK is broadly expressed in many human tissues and cells. It is also overexpressed in many cancers, including colorectal cancer (CRC). Inflammation, as evidenced by inflammatory bowel disease (IBD), is one of the highest risk factors for initiating CRC. This has led to the hypothesis that targeting ILK therapeutically could have potential in CRC, as it regulates different cellular processes associated with CRC development and progression as well as inflammation in the colon. A number of studies have indicated an ILK function in senescence, a cellular process that arrests the cell cycle while maintaining active metabolism and transcription. Senescent cells produce different secretions collectively known as the senescence-associated secretory phenotype (SASP). The SASP secretions influence infiltration of different immune cells, either positively for clearing senescent cells or negatively for promoting tumor growth, reflecting the dual role of senescence in cancer. However, a role for ILK in senescence and immunity in CRC remains to be determined. In this review, we discuss the possible role for ILK in senescence and immunity, paying particular attention to the relevance of ILK in CRC. We also examine how activating Toll-like receptors (TLRs) and their agonists in CRC could trigger immune responses against cancer, as a combination therapy with ILK inhibition.

Substratum stiffness signals through integrin-linked kinase and β1-integrin to regulate midbody proteins and abscission during EMT

Abscission is the final stage of cytokinesis during which the parent cell physically separates to yield two identical daughters. Failure of abscission results in multinucleation, a sign of genomic instability and a precursor to aneuploidy, enabling characteristics of neoplastic progression. Induction of epithelial-mesenchymal transition (EMT) causes multinucleation in mammary epithelial cells cultured on stiff microenvironments that have mechanical properties similar to those found in breast tumors, but not on soft microenvironments reminiscent of the normal mammary gland. Here, we report that on stiff microenvironments, EMT signaling through Snail upregulates the midbody-associated proteins septin-6, Mklp1, and anillin, leading to abscission failure and multinucleation. To uncover the mechanism by which stiff microenvironments promote multinucleation in cells undergoing EMT, we investigated the role of cell-matrix adhesion through β1-integrin and integrin-linked kinase (ILK). We found that ILK expression, but not kinase activity, is required for EMT-associated multinucleation in cells on stiff microenvironments. Conversely, increasing focal adhesions by expressing an autoclustering β1-integrin promotes multinucleation in cells on soft microenvironments. Our data suggest that signaling through focal adhesions causes failure of cytokinesis in cells actively undergoing EMT. These results highlight the importance of tissue mechanics and adhesion in regulating the cellular response to EMT inducers.

Cardiac Differentiation of Human Pluripotent Stem Cells Using Defined Extracellular Matrix Proteins Reveals Essential Role of Fibronectin

Research and therapeutic applications using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) require robust differentiation strategies. Efforts to improve hPSC-CM differentiation have largely overlooked the role of extracellular matrix (ECM). The present study investigates the ability of defined ECM proteins to promote hPSC cardiac differentiation. Fibronectin, laminin-111, and laminin-521 enabled hPSCs to attach and expand; however, fibronectin ECM either endogenously produced or exogenously added promoted, while laminins inhibited, cardiac differentiation in response to growth factors Activin A, BMP4, and bFGF. Inducible shRNA knockdown of fibronectin prevented Brachyury+ mesoderm formation and subsequent hPSC-CM differentiation. Antibodies blocking fibronectin binding to integrin β1, but not α5, inhibited cardiac differentiation. Furthermore, inhibition of integrin-linked kinase blocked cardiac differentiation. These results identify fibronectin, laminin-111 and laminin-521 as defined substrates enabling cardiac differentiation of hPSCs and uncover the essential role of fibronectin and downstream signaling pathways in the early stage of hPSC-CM differentiation.

Inhibition Effect of Chloroquine and Integrin-Linked Kinase Knockdown on Translation in Melanoma Cells

The twofold role of autophagy in cancer is often the therapeutic target. Numerous regulatory pathways are shared between autophagy and other molecular processes needed in tumorigenesis, such as translation or survival signaling. Thus, we have assumed that ILK knockdown should promote autophagy, and used together with chloroquine, an autophagy inhibitor, it could generate a better anticancer effect by dysregulation of common signaling pathways. Expression at the protein level was analyzed using Western Blot; siRNA transfection was done for ILK. Analysis of cell signaling pathways was monitored with phospho-specific antibodies. Melanoma cell proliferation was assessed with the crystal violet test, and migration was evaluated by scratch wound healing assays. Autophagy was monitored by the accumulation of its marker, LC3-II. Our data show that ILK knockdown by siRNA suppresses melanoma cell growth by inducing autophagy through AMPK activation, and simultaneously initiates apoptosis. We demonstrated that combinatorial treatment of melanoma cells with CQ and siILK has a stronger antitumor effect than monotherapy with either of these. It generates the synergistic antitumor effects by the decrease of translation of both global and oncogenic proteins synthesis. In our work, we point to the crosstalk between translation and autophagy regulation.

ATP allosterically stabilizes Integrin-linked kinase for efficient force generation

Focal adhesions link the actomyosin cytoskeleton to the extracellular matrix regulating cell adhesion, shape, and migration. Adhesions are dynamically assembled and disassembled in response to extrinsic and intrinsic forces, but how the essential adhesion component intergrin-linked kinase (ILK) dynamically responds to mechanical force and what role ATP bound to this pseudokinase plays remains elusive. Here, we apply force-probe molecular dynamics simulations of ILK:alpha-parvin coupled to traction force microscopy to explore ILK mechanotransducing functions. We identify two key saltbridge-forming arginines within the allosteric, ATP-dependent force-propagation network of ILK. Disrupting this network by mutation impedes parvin binding, focal adhesion stabilization, force generation, and thus migration. Under tension, ATP shifts the balance from rupture of the complex to protein unfolding, indicating that ATP increases the force threshold required for focal adhesion disassembly. Our study proposes a new role of ATP as an obligatory binding partner for structural and mechanical integrity of the pseudokinase ILK, ensuring efficient cellular force generation and migration.