Rho Kinase Inhibition by AT13148 Blocks Pancreatic Ductal Adenocarcinoma Invasion and Tumor Growth

Abstract

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.

ROCK 2 inhibition triggers the collective invasion of colorectal adenocarcinomas

“…We found that inhibition of ROCK triggers the formation of leaders and the transition from non‐invasive to protrusive cohorts that undergo collective invasion. This is in contradiction with the pro‐invasive role of ROCK identified in CIMP CRCs explants (Zajac et al , ), mice xenografted with cell lines (Itoh et al , ; Croft et al , ; Sadok et al , ) or transgenic models of pancreatic cancer (Rath et al , ). However, our results are in agreement with the recent large‐scale in vivo RNAi screen revealing that loss of Myosin‐II triggers the formation of invasive skin cancers (Schramek et al , ) and that expression of active ROCK2 increases proliferation, but not invasion in similar models (Samuel et al , ).…”

CAF Subpopulations: A New Reservoir of Stromal Targets in Pancreatic Cancer

“…Fasudil, a potent ROCK inhibitor, also reduced PSC activation in a murine model of PDAC, and led to decreased collagen deposition in the TME, increased gemcitabine delivery, and improved survival [111] (Figure 4). Moreover, inhibition of ROCK-based contractility via the small-molecule inhibitor AT13148 resulted in antitumourigenic effects in both in vitro and in vivo models of PDAC (Figure 4) [112]. The same group also established that activation of ROCK in KPC cancer cells promoted protumourigenic ECM remodelling via matrix metalloproteinase (MMP) secretion [113].…”

Targeting the complexity of Src signalling in the tumour microenvironment of pancreatic cancer: from mechanism to therapy

“…Future trials would however need to consider standardisation of the biomarker analysis and interpretation of Merlin loss, sampling of multiple tumour areas where possible to account for potential intratumoural heterogeneity of molecular marker(s) of interest and incorporation of additional promising biomarkers to aid identification of clinical responders to FAK inhibitor-based treatment regimens. Newer ROCK inhibitors (such as ripasudil, CCT129254 or AT13148), are currently being trialled, and utilise a similar 'priming' [92,93] or intermittent regime [201]. Most recently, fasudil, an inexpensive, off-patent ROCK inhibitor, may present a promising new treatment approach for PDAC.…”

“…Src-family kinases (SFKs) not only promote cellmatrix adhesion turnover through FAK, but also regulate Rho family of small GTPases, in particular RhoA and Rac1 activation [85,86]. Rho GTPases are often hijacked by cancers because they regulate diverse cellular processes that are important for tumour growth and metastasis including cytoskeletal dynamics, motility, contractility, cell polarity, membrane transport, gene transcription, as well as regulating the interaction between stromal cells and cancer cells [87][88][89][90][91][92][93]. SFKs control the regulatory molecules of Rho GTPases (guanine nucleotide exchange factors (GEFs), GTPaseactivating proteins (GAPs) and guanine dissociation inhibitors (GDIs)), and it is the tight regulation and extensive crosstalk between Src/FAK and Src/RhoA/ Rac1 that controls integrin-mediated cell adhesion and migration [94][95][96].…”

“…It has recently been shown that using a short-term 'priming' treatment approach to inhibit ROCK signalling can reduce tissue stiffness, improve vascular patency, increase tumour perfusion, decrease in vivo primary tumour growth, metastasis and improve response to standard of care therapy [23,92], similar to chronic fasudil treatment [89]. Newer ROCK inhibitors (such as ripasudil, CCT129254 or AT13148), are currently being trialled, and utilise a similar 'priming' [92,93] or intermittent regime [201]. The rationale behind this novel treatment scheduling involves modulating or 'loosening' the ECM, via ROCK inhibition, prior to chemotherapy administration in order to improve chemotherapy drug perfusion and reduce toxicity [92].…”