ROCK Inhibition Activates MCF-7 Cells

Yang, Seungwon; Kim, Hyun‐Man

doi:10.1371/journal.pone.0088489

Cited by 35 publications

(31 citation statements)

References 53 publications

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“…Interestingly though, Y27632 treatment modestly increased migration and substantially increased invasion of the obscurin knockdown MCF10A cells. This is consistent with previous findings obtained from a variety of cell types for both migration [39, 40] and invasion [41, 42], and likely reflects the fact that complete ROCK inhibition blocks signal inputs from several cellular pathways, while knocking down obscurins has a more modest effect due to its specificity, affecting only RhoA signaling to ROCK.…”

Section: Resultssupporting

confidence: 92%

Loss of the obscurin-RhoGEF downregulates RhoA signaling and increases microtentacle formation and attachment of breast epithelial cells

et al. 2014

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Obscurins are RhoGEF-containing proteins whose downregulation has been implicated in the development and progression of breast cancer. Herein, we aim to elucidate the mechanism for increased motility of obscurin-deficient cells. We show that shRNA-mediated obscurin downregulation in MCF10A cells leads to >50% reduction in RhoA activity relative to scramble control (shCtrl), as well as decreased phosphorylation of RhoA effectors, including myosin light chain phosphatase, myosin light chain, lim kinase, and cofilin, in both attached and suspended cells. These alterations result in decreased actomyosin contractility, allowing suspended cells to escape detachment-induced apoptosis. Moreover, ~40% of shObsc-expressing cells, but only ~10% of shCtrl-expressing cells, extend microtentacles, tubulin-based projections that mediate the attachment of circulating tumor cells to endothelium. Indeed, we show that MCF10A cells expressing shObsc attach in vitro more readily than shCtrl cells, an advantage that persists following taxane exposure. Overall, our data suggest that loss of obscurins may represent a substantial selective advantage for breast epithelial cells during metastasis, and that treatment with paclitaxel may exacerbate this advantage by preferentially allowing obscurin-deficient, stem-like cells to attach to the endothelium of distant sites, a first step towards colonizing metastatic tumors.

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Section: Resultssupporting

confidence: 92%

Loss of the obscurin-RhoGEF downregulates RhoA signaling and increases microtentacle formation and attachment of breast epithelial cells

et al. 2014

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“…As previously reported, MCF7 cells, used here as a positive control for luminal immunostaining, showed activation of a mesenchymal phenotype when treated with ROCKi (Fig. 5d), losing cell-cell contacts and forming many filopodia [32]. MCF10A cells did not appear grossly affected by ROCKi.…”

Section: Primary Dcis Cultures Contain Both Luminal and Basal Mammarysupporting

confidence: 81%

Developing in vitro models of human ductal carcinoma in situ from primary tissue explants

Brown

Dabbs

Lee

et al. 2015

Breast Cancer Res Treat

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Because there are currently no reliable predictors for progression of ductal carcinoma in situ (DCIS) to invasive disease, nearly all patients receive comprehensive therapy, leading to over-treatment in many cases. Few in vitro models for studying DCIS progression have been developed. We report here the successful culture and expansion of primary DCIS from surgical specimens using a conditional reprogramming protocol. Patients with percutaneous core-needle biopsy demonstrating DCIS were enrolled in a tissue banking protocol after informed consent was received. Fresh tissue was taken from lumpectomy or mastectomy specimens, mechanically and enzymatically dissociated, cultured in medium conditioned by irradiated mouse fibroblasts and supplemented with rho-associated protein kinase (ROCK) inhibitor, and characterized by immunocytochemistry. Out of 33 DCIS cases, 58% (19) were expanded for up to 2 months in culture, and 42% (14) were frozen immediately after mechanical dissociation for future growth. The cultures are almost exclusively composed of cytokeratin 8- and EpCAM-positive luminal and cytokeratin 14-, cytokeratin 5-, and p63-positive basal mammary epithelial cells, suggesting maintenance of heterogeneity in vitro. Furthermore, as assessed by luminal and basal marker expression, these cells retain their cellular identities both in the "conditionally reprogrammed" proliferative state and after conditioned media and ROCK inhibitor withdrawal. When grown to 100 % confluency, the cultures organize into luminal and basal layers as well as luminal compartments surrounded by basal cells. Primary cultures of DCIS derived directly from patient tissues can be generated and may serve as in vitro models for the study of DCIS.

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“…38 However, accumulating evidence suggests that therapeutic targeting of ROCK1 may also have a considerable antineoplastic potential. Application of ROCK1 inhibitors reduced the metastatic potential in cells from several kinds of solid tumors, presumably by decreasing cell motility.…”

Section: Discussionmentioning

confidence: 99%

RNAi profiling of primary human AML cells identifies ROCK1 as a therapeutic target and nominates fasudil as an antileukemic drug

Wermke

Camgöz

Paszkowski‐Rogacz

et al. 2015

Blood

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Key Points• Large-scale loss-of-function RNAi screens in patientderived AML cells are feasible and able to pinpoint therapeutic targets.• ROCK1 inhibition exerts antileukemic effects in primary human AML cells in vitro and in vivo.Acute myeloid leukemia (AML) is characterized by a marked genetic heterogeneity, which complicates the development of novel therapeutics. The delineation of pathways essential within an individual patient's mutational background might overcome this limitation and facilitate personalized treatment. We report the results of a large-scale lentiviral lossof-function RNA interference (RNAi) screen in primary leukemic cells. Stringent validation identified 6 genes (BNIPL1, ROCK1, RPS13, STK3, SNX27, WDHD1) whose knockdown impaired growth and viability of the cells. Dependence on these genes was not caused by mutation or overexpression, and although some of the candidates seemed to be rather patient specific, others were essential in cells isolated from other AML patients. In addition to the phenotype observed after ROCK1 knockdown, treatment with the approved ROCK inhibitor fasudil resulted in increased apoptosis and decreased viability of primary AML cells. In contrast to observations in some other malignancies, ROCK1 inhibition did not foster growth of immature malignant progenitors but was toxic to this cell fraction in feeder coculture and xenotransplant experiments, indicating a distinct effect of ROCK1 inhibition on leukemic progenitors. We conclude that large-scale RNAi screens in primary patient-derived cells are feasible and can complement other methods for personalized cancer therapies, such as expression and mutation profiling. (Blood. 2015;125(24):3760-3768)

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ROCK Inhibition Activates MCF-7 Cells

Cited by 35 publications

References 53 publications

Loss of the obscurin-RhoGEF downregulates RhoA signaling and increases microtentacle formation and attachment of breast epithelial cells

Loss of the obscurin-RhoGEF downregulates RhoA signaling and increases microtentacle formation and attachment of breast epithelial cells

Developing in vitro models of human ductal carcinoma in situ from primary tissue explants

RNAi profiling of primary human AML cells identifies ROCK1 as a therapeutic target and nominates fasudil as an antileukemic drug

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