Elucidation of the underlying molecular mechanisms of immune evasion in cancer is critical for the development of immunotherapies aimed to restore and stimulate effective antitumor immunity. Here, we evaluate the role of the actin cytoskeleton in breast cancer cell resistance to cytotoxic natural killer (NK) cells. A significant fraction of breast cancer cells responded to NK-cell attack via a surprisingly rapid and massive accumulation of F-actin near the immunologic synapse, a process we termed “actin response.” Live-cell imaging provided direct evidence that the actin response is associated with tumor cell resistance to NK-cell–mediated cell death. High-throughput imaging flow cytometry analyses showed that breast cancer cell lines highly resistant to NK cells were significantly enriched in actin response-competent cells as compared with susceptible cell lines. The actin response was not associated with a defect in NK-cell activation but correlated with reduced intracellular levels of the cytotoxic protease granzyme B and a lower rate of apoptosis in target cells. Inhibition of the actin response by knocking down CDC42 or N-WASP led to a significant increase in granzyme B levels in target cells and was sufficient to convert resistant breast cancer cell lines into a highly susceptible phenotype. The actin response and its protective effects were fully recapitulated using donor-derived primary NK cells as effector cells. Together, these findings establish the pivotal role of actin remodeling in breast cancer cell resistance to NK-cell–mediated killing.Significance: These findings establish the pivotal role of the actin cytoskeleton in driving breast cancer cell resistance to natural killer cells, a subset of cytotoxic lymphocytes with important roles in innate antitumor immunity. Cancer Res; 78(19); 5631–43. ©2018 AACR.

Recently, a number of two LIM-domain containing proteins (LIMs) have been reported to trigger the formation of actin bundles, a major higher-order cytoskeletal assembly. Here, we analyzed the six Arabidopsis thaliana LIM proteins. Promoter-β-glucuronidase reporter studies revealed that WLIM1, WLIM2a, and WLIM2b are widely expressed, whereas PLIM2a, PLIM2b, and PLIM2c are predominantly expressed in pollen. LIM-green fluorescent protein (GFP) fusions all decorated the actin cytoskeleton and increased actin bundle thickness in transgenic plants and in vitro, although with different affinities and efficiencies. Remarkably, the activities of WLIMs were calcium and pH independent, whereas those of PLIMs were inhibited by high pH and, in the case of PLIM2c, by high [Ca²⁺]. Domain analysis showed that the C-terminal domain is key for the responsiveness of PLIM2c to pH and calcium. Regulation of LIM by pH was further analyzed in vivo by tracking GFP-WLIM1 and GFP-PLIM2c during intracellular pH modifications. Cytoplasmic alkalinization specifically promoted release of GFP-PLIM2c but not GFP-WLIM1, from filamentous actin. Consistent with these data, GFP-PLIM2c decorated long actin bundles in the pollen tube shank, a region of relatively low pH. Together, our data support a prominent role of Arabidopsis LIM proteins in the regulation of actin cytoskeleton organization and dynamics in sporophytic tissues and pollen.

TWISTED DWARF1 determines downstream locations of auxin efflux transporters by adjusting actin cytoskeleton dynamics and mediates NPA action on these in an acti

Hypoxia is a common feature of solid tumours that promotes invasion and metastatic dissemination. Invadopodia are actin-rich membrane protrusions that direct extracellular matrix proteolysis and facilitate tumour cell invasion. Here, we show that CSRP2, an invadopodial actin bundling protein, is upregulated by hypoxia in various breast cancer cell lines, as well as in pre-clinical and clinical breast tumour specimens. We functionally characterized two hypoxia responsive elements within the proximal promoter of CSRP2 gene which are targeted by hypoxia-inducible factor-1 (HIF-1) and required for promoter transactivation in response to hypoxia. Remarkably, CSRP2 knockdown significantly inhibits hypoxia-stimulated invadopodium formation, ECM degradation and invasion in MDA-MB-231 cells, while CSRP2 forced expression was sufficient to enhance the invasive capacity of HIF-1α-depleted cells under hypoxia. In MCF-7 cells, CSRP2 upregulation was required for hypoxia-induced formation of invadopodium precursors that were unable to promote ECM degradation. Collectively, our data support that CSRP2 is a novel and direct cytoskeletal target of HIF-1 which facilitates hypoxia-induced breast cancer cell invasion by promoting invadopodia formation.

We used confocal microscopy and in vitro analyses to show that Nicotiana tabacum WLIM1, a LIM domain protein related to animal Cys-rich proteins, is a novel actin binding protein in plants. Green fluorescent protein (GFP)–tagged WLIM1 protein accumulated in the nucleus and cytoplasm of tobacco BY2 cells. It associated predominantly with actin cytoskeleton, as demonstrated by colabeling and treatment with actin-depolymerizing latrunculin B. High-speed cosedimentation assays revealed the ability of WLIM1 to bind directly to actin filaments with high affinity. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed a highly dynamic in vivo interaction of WLIM1-GFP with actin filaments. Expression of WLIM1-GFP in BY2 cells significantly delayed depolymerization of the actin cytoskeleton induced by latrunculin B treatment. WLIM1 also stabilized actin filaments in vitro. Importantly, expression of WLIM1-GFP in Nicotiana benthamiana leaves induces significant changes in actin cytoskeleton organization, specifically, fewer and thicker actin bundles than in control cells, suggesting that WLIM1 functions as an actin bundling protein. This hypothesis was confirmed by low-speed cosedimentation assays and direct observation of F-actin bundles that formed in vitro in the presence of WLIM1. Taken together, these data identify WLIM1 as a novel actin binding protein that increases actin cytoskeleton stability by promoting bundling of actin filaments.