Non-muscle myosin II (NMII) is reported to play multiple roles during cell migration and invasion. However, the exact biophysical roles of different NMII isoforms during these processes remain poorly ...understood. We analyzed the contributions of NMIIA and NMIIB in three-dimensional (3D) migration and in generating the forces required for efficient invasion by mammary gland carcinoma cells. Using traction force microscopy and microfluidic invasion devices, we demonstrated that NMIIA is critical for generating force during active protrusion, and NMIIB plays a major role in applying force on the nucleus to facilitate nuclear translocation through tight spaces. We further demonstrate that the nuclear membrane protein nesprin-2 is a possible linker coupling NMIIB-based force generation to nuclear translocation. Together, these data reveal a central biophysical role for NMIIB in nuclear translocation during 3D invasive migration, a result with relevance not only to cancer metastasis but for 3D migration in other settings such as embryonic cell migration and wound healing.
Metastatic cancer cells invading through dense tumor stroma experience internal and external forces that are sensed through a variety of mechanosensory proteins that drive adaptations for specific ...environments. Alpha-actinin-4 (ACTN4) is a member of the α-actinin family of actin crosslinking proteins that is upregulated in several types of cancers. It shares 86% protein similarity with α-actinin-1, another non-muscle ACTN isoform, which appears to have a more modest role, if any, in cancer progression. While they share regulatory mechanisms, such as phosphorylation, calcium binding, phosphatidyl inositol binding, and calpain cleavage, α-actinin-4 exhibits a unique mechanosensory regulation that α-actinin-1 does not. This behavior is mediated, at least in part, by each protein’s actin-binding affinity as well as the catch-slip-bond behavior of the actin binding domains. We will discuss currently known modes of ACTN4 regulation, their interactions, and how mechanosensation may provide major therapeutic targeting potential for cancer metastasis.
Non-muscle myosin II (NMII) is a conserved force-producing cytoskeletal enzyme with important but poorly understood roles in cell migration. To investigate myosin heavy chain (MHC) phosphorylation ...roles in 3D migration, we expressed GFP-tagged NMIIA wild-type or mutant constructs in cells depleted of endogenous NMIIA protein. We find that individual mutation or double mutation of Ser-1916 or Ser-1943 to alanine potently blocks recruitment of GFP-NM-IIA filaments to leading edge protrusions in 2D, and this in turn blocks maturation of anterior focal adhesions. When placed in 3D collagen gels, cells expressing wild-type GFP MHC-IIA behave like parental cells, displaying robust and active formation and retraction of protrusions. However, cells depleted of NMIIA or cells expressing the mutant GFP MHC-IIA display severe defects in invasion and in stabilizing protrusions in 3D. These studies reveal an NMIIA-specific role in 3D invasion that requires competence for NMIIA phosphorylation at Ser-1916 and Ser-1943. In sum, these results demonstrate a critical and previously unrecognized role for NMIIA phosphorylation in 3D invasion.
Metastasis is complex, involving multiple genetic, epigenetic, biochemical, and physical changes in the cancer cell and its microenvironment. Cells with metastatic potential are often characterized ...by altered cellular contractility and deformability, lending them the flexibility to disseminate and navigate through different microenvironments. We demonstrate that mechanoresponsiveness is a hallmark of pancreatic cancer cells. Key mechanoresponsive proteins, those that accumulate in response to mechanical stress, specifically nonmuscle myosin IIA (MYH9) and IIC (MYH14), α-actinin 4, and filamin B, were highly expressed in pancreatic cancer as compared with healthy ductal epithelia. Their less responsive sister paralogs-myosin IIB (MYH10), α-actinin 1, and filamin A-had lower expression differential or disappeared with cancer progression. We demonstrate that proteins whose cellular contributions are often overlooked because of their low abundance can have profound impact on cell architecture, behavior, and mechanics. Here, the low abundant protein MYH14 promoted metastatic behavior and could be exploited with 4-hydroxyacetophenone (4-HAP), which increased MYH14 assembly, stiffening cells. As a result, 4-HAP decreased dissemination, induced cortical actin belts in spheroids, and slowed retrograde actin flow. 4-HAP also reduced liver metastases in human pancreatic cancer-bearing nude mice. Thus, increasing MYH14 assembly overwhelms the ability of cells to polarize and invade, suggesting targeting the mechanoresponsive proteins of the actin cytoskeleton as a new strategy to improve the survival of patients with pancreatic cancer. SIGNIFICANCE: This study demonstrates that mechanoresponsive proteins become upregulated with pancreatic cancer progression and that this system of proteins can be pharmacologically targeted to inhibit the metastatic potential of pancreatic cancer cells.
Advanced cancers display cellular heterogeneity driven by self‐renewing, tumorigenic cancer stem cells (CSCs). The use of cell lines to model CSCs is challenging due to the difficulty of identifying ...and isolating cell populations that possess differences in self‐renewal and tumor initiation. To overcome these barriers in triple‐negative breast cancer (TNBC), we developed a CSC system using a green fluorescent protein (GFP) reporter for the promoter of the well‐established pluripotency gene NANOG. NANOG‐GFP+ cells gave rise to both GFP+ and GFP− cells, and GFP+ cells possessed increased levels of the embryonic stem cell transcription factors NANOG, SOX2, and OCT4 and elevated self‐renewal and tumor initiation capacities. GFP+ cells also expressed mesenchymal markers and demonstrated increased invasion. Compared with the well‐established CSC markers CD24−/CD44+, CD49f, and aldehyde dehydrogenase (ALDH) activity, our NANOG‐GFP reporter system demonstrated increased enrichment for CSCs. To explore the utility of this system as a screening platform, we performed a flow cytometry screen that confirmed increased CSC marker expression in the GFP+ population and identified new cell surface markers elevated in TNBC CSCs, including junctional adhesion molecule‐A (JAM‐A). JAM‐A was highly expressed in GFP+ cells and patient‐derived xenograft ALDH+ CSCs compared with the GFP− and ALDH− cells, respectively. Depletion of JAM‐A compromised self‐renewal, whereas JAM‐A overexpression induced self‐renewal in GFP− cells. Our data indicate that we have defined and developed a robust system to monitor differences between CSCs and non‐CSCs in TNBC that can be used to identify CSC‐specific targets for the development of future therapeutic strategies. Stem Cells. Stem Cells 2015;33:2114–2125
Metastases are the cause of the vast majority of cancer deaths. In the metastatic process, cells migrate to the vasculature, intravasate, extravasate, and establish metastatic colonies. This pattern ...of spread requires the cancer cells to change shape and to navigate tissue barriers. Approaches that block this mechanical program represent new therapeutic avenues. We show that 4-hydroxyacetophenone (4-HAP) inhibits colon cancer cell adhesion, invasion, and migration in vitro and reduces the metastatic burden in an in vivo model of colon cancer metastasis to the liver. Treatment with 4-HAP activates nonmuscle myosin-2C (NM2C) (MYH14) to alter actin organization, inhibiting the mechanical program of metastasis. We identify NM2C as a specific therapeutic target. Pharmacological control of myosin isoforms is a promising approach to address metastatic disease, one that may be readily combined with other therapeutic strategies.
Glacial cycles and pre-glacial drainage patterns have imbued the Interior Highlands of the United States with a rich suite of freshwater taxa and phylogroups. However, supplementation of sportfish ...from the Great Lakes into the waterbodies of this region, including the Missouri, Ouachita, Black, White, and Little Red River drainages, may have obscured phylogeographic patterns. Walleye (
Sander vitreus
), one of the most common sportfish in the eastern US, inhabit the Interior Highlands but their population genetic composition and structure in this region has received little attention. We examined the genetic composition of walleye (
n
= 643) in the Interior Highlands using microsatellite markers and a subsample (
n
= 188) with mitochondrial DNA and found significant genetic differences among walleye in our study area. Walleye from the Missouri, Ouachita, White, and Little Red drainages were most closely related to a Great Lakes reference sample, a common stocking source. However, the Black River, in the easternmost portion of the Interior Highlands, contained walleye with mitochondrial DNA that was closely related to walleye from the Eastern Highlands. The remainder of the study area drainages contained a mix of walleye groups, more closely related to Great Lakes rather than Highlands walleye but not definitively the product of stocking. Though managers have relied on mitochondrial markers for stock identification in the past, we recommend that walleye in regions receiving little research attention be analyzed with nuclear markers to better understand and preserve genetic diversity and that managers stock with local walleye only within the drainages that we identified as genetically distinct: Black, Missouri, and White/Little Red/Ouachita.
The Coronary Artery Disease Reporting and Data System (CAD-RADS) provides a lexicon and standardized reporting system for coronary CT angiography.
To evaluate inter-observer agreement of the CAD-RADS ...among an panel of early career and expert readers.
Four early career and four expert cardiac imaging readers prospectively and independently evaluated 50 coronary CT angiography cases using the CAD-RADS lexicon. All readers assessed image quality using a five-point Likert scale, with mean Likert score ≥4 designating high image quality, and <4 designating moderate/low image quality. All readers were blinded to medical history and invasive coronary angiography findings. Inter-observer agreement for CAD-RADS assessment categories and modifiers were assessed using intra-class correlation (ICC) and Fleiss' Kappa (κ).The impact of reader experience and image quality on inter-observer agreement was also examined.
Inter-observer agreement for CAD-RADS assessment categories was excellent (ICC 0.958, 95% CI 0.938–0.974, p < 0.0001). Agreement among expert readers (ICC 0.925, 95% CI 0.884–0.954) was marginally stronger than for early career readers (ICC 0.904, 95% CI 0.852–0.941), both p < 0.0001. High image quality was associated with stronger agreement than moderate image quality (ICC 0.944, 95% CI 0.886–0.974 vs. ICC 0.887, 95% CI 0.775–0.95, both p < 0.0001). While excellent inter-observer agreement was observed for modifiers S (stent) and G (bypass graft) (both κ = 1.0), only fair agreement (κ = 0.40) was observed for modifier V (high risk plaque).
Inter-observer reproducibility of CAD-RADS assessment categories and modifiers is excellent, except for high-risk plaque (modifier V) which demonstrates fair agreement. These results suggest CAD-RADS is feasible for clinical implementation.
Cardiovascular computed tomography (CCT) is a well-validated non-invasive imaging tool with an ever-expanding array of applications beyond the assessment of coronary artery disease. These include the ...evaluation of structural heart diseases, congenital heart diseases, peri-procedural electrophysiology applications, and the functional evaluation of ischemia. This breadth requires a robust and diverse training curriculum to ensure graduates of CCT training programs meet minimum competency standards for independent CCT interpretation. This statement from the Society of Cardiovascular Computed Tomography aims to supplement existing societal training guidelines by providing a curriculum and competency framework to inform the development of a comprehensive, integrated training experience for cardiology and radiology trainees in CCT.
Cardiovascular computed tomography (CCT) is a well-validated non-invasive imaging tool with an ever-expanding array of applications beyond the assessment of coronary artery disease. These include the ...evaluation of structural heart diseases, congenital heart diseases, peri-procedural electrophysiology applications, and the functional evaluation of ischemia. This breadth requires a robust and diverse training curriculum to ensure graduates of CCT training programs meet minimum competency standards for independent CCT interpretation. This statement from the Society of Cardiovascular Computed Tomography aims to supplement existing societal training guidelines by providing a curriculum and competency framework to inform the development of a comprehensive, integrated training experience for cardiology and radiology trainees in CCT.
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