ROCK signalling induced gene expression changes in mouse pancreatic ductal adenocarcinoma cells

The RhoA and RhoC GTPases act via the ROCK1 and ROCK2 kinases to promote actomyosin contraction, resulting in directly induced changes in cytoskeleton structures and altered gene transcription via several possible indirect routes. Elevated activation of the Rho/ROCK pathway has been reported in several diseases and pathological conditions, including disorders of the central nervous system, cardiovascular dysfunctions and cancer. To determine how increased ROCK signalling affected gene expression in pancreatic ductal adenocarcinoma (PDAC) cells, we transduced mouse PDAC cell lines with retroviral constructs encoding fusion proteins that enable conditional activation of ROCK1 or ROCK2, and subsequently performed RNA sequencing (RNA-Seq) using the Illumina NextSeq 500 platform. We describe how gene expression datasets were generated and validated by comparing data obtained by RNA-Seq with RT-qPCR results. Activation of ROCK1 or ROCK2 signalling induced significant changes in gene expression that could be used to determine how actomyosin contractility influences gene transcription in pancreatic cancer

Targeting ROCK activity to disrupt and prime pancreatic cancer for chemotherapy

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease; the identification of novel targets and development of effective treatment strategies are urgently needed to improve patient outcomes. Remodeling of the pancreatic stroma occurs during PDAC development, which drives disease progression and impairs responses to therapy. The actomyosin regulatory ROCK1 and ROCK2 kinases govern cell motility and contractility, and have been suggested to be potential targets for cancer therapy, particularly to reduce the metastatic spread of tumor cells. However, ROCK inhibitors are not currently used for cancer patient treatment, largely due to the overwhelming challenge faced in the development of anti-metastatic drugs, and a lack of clarity as to the cancer types most likely to benefit from ROCK inhibitor therapy. In 2 recent publications, we discovered that ROCK1 and ROCK2 expression were increased in PDAC, and that increased ROCK activity was associated with reduced survival and PDAC progression by enabling extracellular matrix (ECM) remodeling and invasive growth of pancreatic cancer cells. We also used intravital imaging to optimize ROCK inhibition using the pharmacological ROCK inhibitor fasudil (HA-1077), and demonstrated that short-term ROCK targeting, or ‘priming’, improved chemotherapy efficacy, disrupted cancer cell collective movement, and impaired metastasis. This body of work strongly indicates that the use of ROCK inhibitors in pancreatic cancer therapy as ‘priming’ agents warrants further consideration, and provides insights as to how transient mechanical manipulation, or fine-tuning the ECM, rather than chronic stromal ablation might be beneficial for improving chemotherapeutic efficacy in the treatment of this deadly disease

A one-step procedure to probe the viscoelastic properties of cells by Atomic Force Microscopy

The increasingly recognised importance of viscoelastic properties of cells in pathological conditions requires rapid development of advanced cell microrheology technologies. Here, we present a novel Atomic Force Microscopy (AFM)-microrheology (AFM2) method for measuring the viscoelastic properties in living cells, over a wide range of continuous frequencies (0.005 Hz ~ 200 Hz), from a simple stress-relaxation nanoindentation. Experimental data were directly analysed without the need for pre-conceived viscoelastic models. We show the method had an excellent agreement with conventional oscillatory bulk-rheology measurements in gels, opening a new avenue for viscoelastic characterisation of soft matter using minute quantity of materials (or cells). Using this capability, we investigate the viscoelastic responses of cells in association with cancer cell invasive activity modulated by two important molecular regulators (i.e. mutation of the p53 gene and Rho kinase activity). The analysis of elastic (G′(ω)) and viscous (G″(ω)) moduli of living cells has led to the discovery of a characteristic transitions of the loss tangent (G″(ω)/G′(ω)) in the low frequency range (0.005 Hz ~ 0.1 Hz) that is indicative of the capability for cell restructuring of F-actin network. Our method is ready to be implemented in conventional AFMs, providing a simple yet powerful tool for measuring the viscoelastic properties of living cells

Tissue-selective expression of a conditionally-active ROCK2-estrogen receptor fusion protein

The serine/threonine kinases ROCK1 and ROCK2 are central mediators of actomyosin contractile force generation that act downstream of the RhoA small GTP-binding protein. As a result, they have key roles in regulating cell morphology and proliferation, and have been implicated in numerous pathological conditions and diseases including hypertension and cancer. Here we describe the generation of a gene-targeted mouse line that enables CRE-inducible expression of a conditionally-active fusion between the ROCK2 kinase domain and the hormone-binding domain of a mutated estrogen receptor (ROCK2:ER). This two-stage system of regulation allows for tissue-selective expression of the ROCK2:ER fusion protein, which then requires administration of estrogen analogues such as tamoxifen or 4-hydroxytamoxifen to elicit kinase activity. This conditional gain-of-function system was validated in multiple tissues by crossing with mice expressing CRE recombinase under the transcriptional control of cytokeratin14 (K14), murine mammary tumor virus (MMTV) or cytochrome P450 Cyp1A1 (Ah) promoters, driving appropriate expression in the epidermis, mammary or intestinal epithelia respectively. Given the interest in ROCK signaling in normal physiology and disease, this mouse line will facilitate research into the consequences of ROCK activation that could be used to complement conditional knockout models

Regulation of pancreatic cancer aggressiveness by stromal stiffening

No abstract available

ROCK signaling promotes collagen remodeling to facilitate invasive pancreatic ductal adenocarcinoma tumor cell growth

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer death; identifying PDAC enablers may reveal potential therapeutic targets. Expression of the actomyosin regulatory ROCK1 and ROCK2 kinases increased with tumor progression in human and mouse pancreatic tumors, while elevated ROCK1/ROCK2 expression in human patients, or conditional ROCK2 activation in a KrasG12D/p53R172H mouse PDAC model, was associated with reduced survival. Conditional ROCK1 or ROCK2 activation promoted invasive growth of mouse PDAC cells into three‐dimensional collagen matrices by increasing matrix remodeling activities. RNA sequencing revealed a coordinated program of ROCK‐induced genes that facilitate extracellular matrix remodeling, with greatest fold‐changes for matrix metalloproteinases (MMPs) Mmp10 and Mmp13. MMP inhibition not only decreased collagen degradation and invasion, but also reduced proliferation in three‐dimensional contexts. Treatment of KrasG12D/p53R172H PDAC mice with a ROCK inhibitor prolonged survival, which was associated with increased tumor‐associated collagen. These findings reveal an ancillary role for increased ROCK signaling in pancreatic cancer progression to promote extracellular matrix remodeling that facilitates proliferation and invasive tumor growth

Empfehlungen zu einem Inklusionskonzept am Oberstufen-Kolleg Bielefeld: Ein Diskussionspapier der FEP-Gruppe „Kernaufgabe Inklusion am Oberstufen-Kolleg“

Das Oberstufen-Kolleg Bielefeld verfügt über eine langjährige Erfahrung im Umgang mit seiner heterogenen Kollegiatenschaft und den daraus resultierenden unterschiedlichen pädagogischen Bedürfnissen. Die Projektgruppe „KIOSK – Kernaufgabe Inklusion am Oberstufen-Kolleg“ hat während der dreijährigen Projektlaufzeit von 2014 bis 2017 das Anliegen verfolgt, auf die Erfahrungen des Hauses in der Arbeit mit der heterogenen Kollegiatenschaft aufzubauen und systematischere Strukturen zu schaffen, die es ermöglichen, näher auf die individuellen pädagogischen Bedürfnisse der Kollegiat*innen in unterschiedlichen Bereichen des Schullebens eingehen zu können. Zu diesem Zwecke wurden mit unterschiedlichen Akteursgruppen Interviews und Gruppendiskussionen geführt, schulische Strukturen und Prozesse mit inklusionssensiblem Blick analysiert und z.T. auch adaptiert, neue Kursformate erprobt und Transferveranstaltungen durchgeführt. Die Erkenntnisse dieser umfassenden Analyse sind im vorliegenden Papier in Form von Empfehlungen zu einem Inklusionskonzept am Oberstufen-Kolleg Bielefeld gebündelt, die sich insgesamt auf zwölf unterschiedliche Be­reiche beziehen. Einige dieser Bereiche betreffen die Beratungs- und Unterstützungsstrukturen für Kollegiat*innen (etwa hinsichtlich ihrer Potenzialförderung, sonderpädagogischer Expertise, Schulsozialarbeit und Coaching-Programmen); andere Bereiche beziehen sich auf Strukturen und Prozesse des Hauses (z.B. das Aufnahmeverfahren, die Gewährung von Nachteils­ausgleich und der Umgang mit psychisch erkrankten Kollegiat*innen). Obwohl sich das vorliegende Dokument in erster Linie als Arbeitspapier für die weitere inklusive Schulentwicklung des Oberstufen-Kollegs versteht, mag es darüber hinaus jedoch trotz seiner Spezifik auch wertvoll für andere (Oberstufen-)Schulen sein – etwa, um den Blick für inklusive Strukturen und Prozesse zu schärfen, mögliche blinde Flecken aufzuspüren und Impulse für die eigene Schulentwicklung zu bekommen

Epidermal ROCK2-induces AKT1/GSK3β/β-catenin, NFκB and dermal tenascin-C; but enhanced differentiation and p53/p21 inhibit papilloma

ROCK2 roles in epidermal differentiation and carcinogenesis have been investigated in mice expressing an RU486-inducible, 4HT-activated ROCK2 transgene (K14.creP/lslROCKer). RU486/4HT-mediated ROCKer activation induced epidermal hyperplasia similar to cutaneous oncogenic rasHa (HK1.ras); however ROCKer did not elicit papillomas. Instead, anomalous basal-layer ROCKer expression corrupted normal ROCK2 roles underlying epidermal rigidity/stiffness and barrier maintanance, resulting in premature keratin K1, loricrin and filaggrin expression. Also, hyperproliferative/stress-associated keratin K6 was reduced; possibly reflecting altered ROCK2 roles in epidermal rigidity and keratinocyte flexibility/migration during wound healing. Consistent with increased proliferation, K14.creP/lslROCKer hyperplasia displayed supra-basal-to-basal increases in activated p-AKT1, inactivated p-GSK3β ser9 and membranous/nuclear β-catenin expression together with weak NFκB, which were absent in equivalent HK1.ras hyperplasia. Furthermore, ROCKer-mediated increases in epidermal rigidity via p-MypT1 inactivation/elevated MLC, coupled to anomalous β-catenin expression, induced tenascin C-positive dermal fibroblasts. Alongside an altered ECM, these latent tenascin C-positive dermal fibroblasts may become putative pre-cancer–associated fibroblasts (pre-CAFs) and establish a susceptibility that subsequently contributes to tumour progression. However, anomalous differentiation was also accompanied by an immediate increase in basal-layer p53/p21 expression; suggesting that while ROCK2/AKT1/β-catenin activation increased keratinocyte proliferation resulting in hyperplasia, compensatory p53/p21 and accelerated differentiation helped inhibit papillomatogenesis

Rho kinase inhibition by AT13148 blocks pancreatic ductal adenocarinoma invasion and tumor growth

The high mortality of pancreatic cancer demands that new therapeutic avenues be developed. The orally available small molecule inhibitor AT13148 potently inhibits ROCK1 and ROCK2 kinases that regulate the actomyosin cytoskeleton. We previously reported that ROCK kinase expression increases with human and mouse pancreatic cancer progression and that conditional ROCK activation accelerates mortality in a genetically modified LSL-KrasG12D; LSL-p53R172H; Pdx1-Cre; (KPC) mouse pancreatic cancer model. In this study, we show that treatment of KPC mouse and human TKCC5 patient-derived pancreatic tumor cells with AT13148, as well as the ROCK selective inhibitors Y27632 and H1152, act comparably in blocking ROCK substrate phosphorylation. AT13148, Y27632, and H1152 induced morphological changes and reduced cellular contractile force generation, motility on pliable discontinuous substrates, and three-dimensional collagen matrix invasion. AT13148 treatment reduced subcutaneous tumor growth and blocked invasion of healthy pancreatic tissue by KPC tumor cells in vivo without affecting proliferation, suggesting a role for local tissue invasion as a contributor to primary tumor growth. These results suggest that AT13148 has anti-tumor properties that may be beneficial in combination therapies or in the adjuvant setting to reduce pancreatic cancer cell invasion and slow primary tumor growth. AT13148 might also have the additional benefit of enabling tumor resection by maintaining separation between tumor and healthy tissue boundaries