Cancer-associated fibroblasts (CAFs) are activated fibroblasts that constitute the major components of tumor microenvironment (TME) and play crucial roles in tumor development and metastasis. Here, ...we generated fibroblast-specific inducible focal adhesion kinase (FAK) knockout (cKO) mice in a breast cancer model to study potential role and mechanisms of FAK signaling in CAF to promote breast cancer metastasis in vivo. While not affecting primary tumor development and growth, FAK deletion significantly suppressed breast cancer metastasis in vivo. Analyses of CAFs derived from cKO mice as well as human CAFs showed that FAK is required for their activity to promote mammary tumor cell migration. We further showed that FAK ablation in CAFs decreased exosome functions to promote tumor cell migration and other activities, which could contribute to the reduced metastasis observed in cKO mice. Lastly, profiling of miRs from CAF exosomes showed alterations of several exosomal miRs in FAK-null CAFs, and further analysis suggested that miR-16 and miR-148a enriched in exosomes from FAK-null CAFs contribute to the reduced tumor cell activities and metastasis. Together, these results identify a new role for FAK signaling in CAFs that regulate their intercellular communication with tumor cells to promote breast cancer metastasis.
Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays critical roles in integrin-mediated signal transductions and also participates in signaling by other cell surface receptors. In ...integrin-mediated cell adhesion, FAK is activated via disruption of an auto-inhibitory intra-molecular interaction between its amino terminal FERM domain and the central kinase domain. The activated FAK forms a complex with Src family kinases, which initiates multiple downstream signaling pathways through phosphorylation of other proteins to regulate different cellular functions. Multiple downstream signaling pathways are identified to mediate FAK regulation of migration of various normal and cancer cells. Extensive studies in cultured cells as well as conditional FAK knockout mouse models indicated a critical role of FAK in angiogenesis during embryonic development and cancer progression. More recent studies also revealed kinase-independent functions for FAK in endothelial cells and fibroblasts. Consistent with its roles in cell migration and angiogenesis, increased expression and/or activation of FAK are found in a variety of human cancers. Therefore, small molecular inhibitors for FAK kinase activity as well as future development of novel therapies targeting the potentially kinase-independent functions of FAK are promising treatments for metastatic cancer as well as other diseases.
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Combining luminescent transition metal complex with super‐resolution microscopy is an excellent strategy for the long‐term visualization of the dynamics of subcellular structures in living cells. ...However, it remains unclear whether iridium(III) complexes are applicable for a particular type of super‐resolution technique, structured illumination microscopy (SIM), to image subcellular structures. Herein, an iridium(III) dye, to track mitochondrial dynamics in living cells under SIM is described. The dye demonstrates excellent specificity and photostability and satisfactory cell permeability. While using SIM to image mitochondria, an ≈80 nm resolution is achieved that allows the clear observation of the structure of mitochondrial cristae. The dye is used to monitor and quantify mitochondrial dynamics relative to lysosomes, including fusion involved in mitophagy, and newly discovered mitochondria–lysosome contact (MLC) under different conditions. The MLC remains intact and fusion vanishes when five receptors, p62, NDP52, OPTN, NBR1, and TAX1BP1, are knocked out, suggesting that these two processes are independent.
A novel iridium(III) luminophore can track mitochondrial dynamics under structured illumination microscopy up to ≈80 nm resolution. This luminophore can assist researchers in quantifying and understanding the formation and function of the interactive behavior between mitochondria and lysosomes.
Breast cancer is a heterogeneous disease, and stratification of tumors is paramount to achieve better clinical outcomes. While it is common to stratify and treat breast tumors as a single entity, ...insights from studies on intratumoral heterogeneity and cancer stem cells raise the possibility that multiple breast cancer subtypes may coexist within a tumor. A role for plasticity in driving dynamic conversions between breast cancer subtypes is proposed, and the clinical implications include a need for combinatorial therapeutic strategies that account for the discrete disease entities and their plasticity. Accordingly, the advent of single-cell technologies will be crucial in enabling the diagnosis and stratification of distinct disease subtypes down to the cellular level.
Cellular interactions with extracellular matrix play essential roles in tumor initiation, progression and metastasis. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase identified as a key ...mediator of signaling by integrins, a major family of cell surface receptors for extracellular matrix, as well as other receptors in both normal and cancer cells. FAK is activated by integrins through disruption of an auto-inhibitory intra-molecular interaction between its kinase domain and the amino terminal FERM domain. The activated FAK forms a binary complex with Src family kinases which can phosphorylate other substrates and trigger multiple intracellular signaling pathways to regulate various cellular functions. Subcellular localization of FAK in focal adhesions is essential for FAK signaling, which is another distinguishing feature of the kinase. Integrin-FAK signaling has been shown to activate a number of signaling pathways through phosphorylation and protein-protein interactions to promote tumorigenesis. FAK also plays a prominent role in tumor progression and metastasis through its regulation of both cancer cells and their microenvironments including cancer cell migration, invasion, epithelial to mesenchymal transition, and angiogenesis. More recently, a role for FAK in tumor initiation and progression has been demonstrated directly using xenograft as well as conditional knockout mouse models. In agreement with these experimental data, overexpression and activation of FAK have been found in a variety of human cancers. A number of small molecule inhibitors for FAK have been developed and in various phases of testing for cancer treatments. Overall, the intensive research on FAK signaling in cancer have yielded a wealth of information on this pivotal kinase and these and future studies are leading to potentially novel therapies for cancer.
Abstract Focal adhesion kinase (FAK) is an intracellular non-receptor tyrosine kinase. In addition to its role as a major mediator of signal transduction by integrins, FAK also participates in ...signaling by a wide range of extracellular stimuli including growth factors, G-protein-coupled receptor agonists, cytokines, and other inflammatory mediators. The link between FAK and breast cancers is strongly suggested by a number of reports showing that FAK gene is amplified and overexpressed in a large fraction of breast cancer specimens. In addition, increased FAK expression and activity frequently correlate with metastatic disease and poor prognosis. Since its discovery in early 1990s, numerous studies have shown a role for FAK in the regulation of cell spreading, adhesion, migration, survival, proliferation, differentiation, and angiogenesis. Many of these studies in cultured cells provided strong evidence to connect FAK expression/activation to the promotion of cancer. Recently, a prominent role of FAK in promoting mammary tumorigenesis, progression and metastasis has been unveiled by different animal models of human breast cancer, including xenograft models in immunodeficient rodents and spontaneous tumor models in transgenic mice that have specific deletion of FAK in the mammary epithelial cells during embryonic or postnatal development. These in vivo studies established FAK as a prominent determinant in mammary cancer initiation, progression and metastasis. Furthermore, a novel function of FAK in maintaining mammary cancer stem/progenitor cells in vivo has been recently reported, which may provide a novel cellular mechanism of FAK in promoting breast cancer initiation and progression. The wealth of knowledge accumulated over almost two decades of research on FAK should help to design potentially novel therapies for breast cancer.
Super-resolution optical microscopy has extended the spatial resolution of cell biology from the cellular level to the nanoscale, enabling the observation of the interactive behavior of single ...mitochondria and lysosomes. Quantitative parametrization of interactions between mitochondria and lysosomes under super-resolution optical microscopy, however, is currently unavailable, which has severely limited our understanding of the molecular machinery underlying mitochondrial functionality. Here, we introduce an M-value to quantitatively investigate mitochondria and lysosome contact (MLC) and mitophagy under structured illumination microscopy. We found that the M-value for an MLC is typically less than 0.4, whereas in mitophagy it ranges from 0.5 to 1.0. This system permits further investigation of the detailed molecular mechanism governing the interactive behavior of mitochondria and lysosomes.
Autophagy represents a fundamental mechanism for maintaining cell survival and tissue homeostasis in response to physiological and pathological stress. Autophagy initiation converges on the ...FIP200‐ATG13‐ULK1 complex wherein the serine/threonine kinase ULK1 plays a central role. Here, we reveal that the E3 ubiquitin ligase TRIM27 functions as a negative regulatory component of the FIP200‐ATG13‐ULK1 complex. TRIM27 directly polyubiquitinates ULK1 at K568 and K571 sites with K48‐linked ubiquitin chains, with proteasomal turnover maintaining control over basal ULK1 levels. However, during starvation‐induced autophagy, TRIM27 catalyzes non‐degradative K6‐ and K11‐linked ubiquitination of the serine/threonine kinase 38‐like (STK38L) kinase. In turn, STK38L ubiquitination promotes its activation and phosphorylation of ULK1 at Ser495, rendering ULK1 in a permissive state for TRIM27‐mediated hyper‐ubiquitination of ULK1. This cooperative mechanism serves to restrain the amplitude and duration of autophagy. Further evidence from mouse models shows that basal autophagy levels are increased in Trim27 knockout mice and that Trim27 differentially regulates tumorigenesis and metastasis. Our study identifies a key role of STK38L‐TRIM27‐ULK1 signaling axis in negatively controlling autophagy with relevance established in human breast cancer.
Synopsis
While ULK1 is essential for autophagy initiation, the mechanisms controlling its expression levels and function remain unclear. This report discloses how E3 ubiquitin ligase TRIM27 suppresses ULK1 protein expression to restrain autophagy through dual ubiquitination events, differentially impacting tumorigenesis.
E3 ligase TRIM27 negatively regulates autophagy flux under basal and starvation conditions.
TRIM27 mediates ubiquitination of ULK1 via K48‐linked chains, inducing its proteasomal degradation.
Starvation induces TRIM27 ubiquitination and activation of STK38L kinase via K6‐linked chains.
STK38L phosphorylates ULK1, providing a permissive state for increased ULK1 ubiquitination by TRIM27.
Ablation of TRIM27 in the PyMT model inhibits murine mammary tumor initiation but promotes metastasis.
E3 ligase TRIM27 and STK38L kinase synergistically control autophagy acting as a rheostat on ULK1.
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