Activation of the adaptive Ire1-XBP1 pathway has been identified in many solid tumors and hematologic malignancies, including multiple myeloma (MM). Here, we report the identification of STF-083010, a novel small-molecule inhibitor of Ire1. STF-083010 inhibited Ire1 endonuclease activity, without affecting its kinase activity, after endoplasmic reticulum stress both in vitro and in vivo. Treatment with STF-083010 showed significant antimy-eloma activity in model human MM xenografts. Similarly, STF-083010 was preferentially toxic to freshly isolated human CD138(+) MM cells compared with other similarly isolated cell populations. The identification of this novel Ire1 inhibitor supports the hypothesis that the Ire1-XBP1 axis is a promising target for anticancer therapy, especially in the context of MM

Evaluative conditioning (EC) refers to the formation or change of an attitude towards an object following that object's pairing with positively or negatively valenced stimuli. We provide evidence that EC can occur through an implicit misattribution mechanism in which an evaluative response evoked by a valenced stimulus is incorrectly and implicitly attributed to another stimulus, forming or changing an attitude towards this other stimulus. Five studies measured or manipulated variables related to the potential for the misattribution of an evaluation, or "source confusability." Greater EC was observed when participants' eye gaze shifted frequently between a valenced and neutral stimulus (Studies 1 & 2), when the two stimuli appeared in close spatial proximity (Study 3), and when the neutral stimulus was made more perceptually salient than the valenced stimulus due to its larger size (Study 4). In other words, conditions conducive to source confusability increased EC. Study 5 provided evidence for multiple mechanisms of EC by comparing the effects of mildly evocative valenced stimuli (those evoking responses that might more easily be misattributed to another object) to more strongly evocative stimuli.

Hypoxia is a dynamic feature of the tumor microenvironment that contributes to drug resistance and cancer progression. We previously showed that components of the Unfolded Protein Response (UPR) elicited by endoplasmic reticulum (ER) stress, are also activated by hypoxia in vitro and in animal and human patient tumors. Here, we report that ER stressors, such as thapsigargin or the clinically used proteasome inhibitor bortezomib, exhibit significantly higher cytotoxicity towards hypoxic compared to normoxic tumor cells, which is accompanied by enhanced activation of UPR effectors in vitro and UPR reporter activity in vivo. Treatment of cells with the translation inhibitor cycloheximide, which relieves ER load, ameliorated this enhanced cytotoxicity, indicating that the increased cytotoxicity is ER stress-dependent. The mode of cell death was cell-type dependent, since DLD1 colorectal carcinoma cells exhibited enhanced apoptosis, whereas in HeLa cervical carcinoma cells, activated autophagy blocked apoptosis and eventually led to necrosis. Pharmacologic or genetic ablation of autophagy increased the levels of apoptosis. These results demonstrate that hypoxic tumor cells, which are generally more resistant to genotoxic agents, are hypersensitive to proteasome inhibitors and suggest that combining bortezomib with therapies that target the normoxic fraction of human tumors can lead to more effective tumor control.

Cancer cells are softer than the normal cells, and metastatic cells are even softer. These changes in biomechanical properties contribute to cancer progression by facilitating cell movement through physically constraining environments. To identify properties that enabled passage through physical constraints, cells that were more efficient at moving through narrow membrane micropores were selected from established cell lines. By examining micropore-selected human MDA MB 231 breast cancer and MDA MB 435 melanoma cancer cells, membrane fluidity and nuclear elasticity were excluded as primary contributors. Instead, reduced actin cytoskeleton anisotropy, focal adhesion density and cell stiffness were characteristics associated with efficient passage through constraints. By comparing transcriptomic profiles between the parental and selected populations, increased Ras/MAPK signalling was linked with cytoskeleton rearrangements and cell softening. MEK inhibitor treatment reversed the transcriptional, cytoskeleton, focal adhesion and elasticity changes. Conversely, expression of oncogenic KRas in parental MDA MB 231 cells, or oncogenic BRaf in parental MDA MB 435 cells, significantly reduced cell stiffness. These results reveal that MAPK signalling, in addition to tumour cell proliferation, has a significant role in regulating cell biomechanics. .

Apoptosis is characterized by profound morphological changes, but their physiological purpose is unknown. To characterize the role of apoptotic cell contraction, ROCK1 was rendered caspase non-cleavable (ROCK1nc) by mutating Aspartate 1113, which revealed that ROCK1 cleavage was necessary for forceful contraction and membrane blebbing. When homozygous ROCK1nc mice were treated with the liver-selective apoptotic stimulus of diethylnitrosamine, ROCK1nc mice had more profound liver damage with greater neutrophil infiltration than wild-type mice. Inhibition of the damage associated molecular pattern protein HMGB1 or signalling by its cognate receptor TLR4 lowered neutrophil infiltration and reduced liver damage. ROCK1nc mice also developed fewer diethylnitrosamine-induced hepatocellular carcinoma (HCC) tumours, while HMGB1 inhibition increased HCC tumour numbers. Thus, ROCK1 activation and consequent cell contraction are required to limit sterile inflammation and damage amplification following tissue-scale cell death. Additionally, these findings reveal a previously unappreciated role for acute sterile inflammation as an efficient tumour suppressive mechanism.

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.

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The myotonic dystrophy-related Cdc42-binding kinases MRCKα and MRCKβ contribute to the regulation of actin-myosin cytoskeleton organization and dynamics, acting in concert with the Rho-associated coiled-coil kinases ROCK1 and ROCK2. The absence of highly potent and selective MRCK inhibitors has resulted in relatively little knowledge of the potential roles of these kinases in cancer. Here, we report the discovery of the azaindole compounds BDP8900 and BDP9066 as potent and selective MRCK inhibitors that reduce substrate phosphorylation, leading to morphologic changes in cancer cells along with inhibition of their motility and invasive character. In over 750 human cancer cell lines tested, BDP8900 and BDP9066 displayed consistent antiproliferative effects with greatest activity in hematologic cancer cells. Mass spectrometry identified MRCKα S1003 as an autophosphorylation site, enabling development of a phosphorylation-sensitive antibody tool to report on MRCKα status in tumor specimens. In a two-stage chemical carcinogenesis model of murine squamous cell carcinoma, topical treatments reduced MRCKα S1003 autophosphorylation and skin papilloma outgrowth. In parallel work, we validated a phospho-selective antibody with the capability to monitor drug pharmacodynamics. Taken together, our findings establish an important oncogenic role for MRCK in cancer, and they offer an initial preclinical proof of concept for MRCK inhibition as a valid therapeutic strategy. The development of selective small-molecule inhibitors of the Cdc42-binding MRCK kinases reveals their essential roles in cancer cell viability, migration, and invasive character. .