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Increased GBM cell invasion in vivo is confirmed by a human specific antibody against HLA and measurement of tumour volume by multi-slice T2 MR imaging

GFP E2 cells irradiated with 2Gy in an ex vivo brain slice motility assay

Supplemental Figure 1. Kinome selectivity profiles for BDP8900 and BDP9066. Supplemental Figure 2. Cancer cell line sensitivity to BDP8900 and BDP9066. Supplemental Figure 3. MRCKα phosphorylations compared with MRCKβ. Supplemental Figure 4. MRCKα, MRCKβ and MRCKα pS1003 antibody validation. Supplemental Figure 5. MRCK and DMPK expression in MDA MB 231 D3H2 LN human breast cancer cells, SCC12 human squamous cell carcinoma cells, and human skeletal muscle. Supplemental Table 1. Crystallographic data and model statistics. Supplemental Table 2: BDP8900 and BDP9066 selectivity in vitro (in-house determinations). Supplemental Table 3. Kinase selectivity screen for BDP8900 and BDP9066. Supplemental Table 4. BDP8900 selectivity in vitro (out-sourced determinations). Supplemental Table 5. BDP9066 selectivity in vitro (out-sourced determinations). Supplemental Table 6. Cancer cell line EC50 values for BDP8900 and BDP9066. Supplemental Table 7. MRCKα phosphorylation sites. Supplemental Table 8. MRCKα phosphorylation on S1003.

Glioblastoma (GBM) is an aggressive and incurable primary brain tumor that causes severe neurologic, cognitive, and psychologic symptoms. Symptoms are caused and exacerbated by the infiltrative properties of GBM cells, which enable them to pervade the healthy brain and disrupt normal function. Recent research has indicated that although radiotherapy (RT) remains the most effective component of multimodality therapy for patients with GBM, it can provoke a more infiltrative phenotype in GBM cells that survive treatment. Here, we demonstrate an essential role of the actin-myosin regulatory kinase myotonic dystrophy kinase-related CDC42-binding kinase (MRCK) in mediating the proinvasive effects of radiation. MRCK-mediated invasion occurred via downstream signaling to effector molecules MYPT1 and MLC2. MRCK was activated by clinically relevant doses per fraction of radiation, and this activation was concomitant with an increase in GBM cell motility and invasion. Furthermore, ablation of MRCK activity either by RNAi or by inhibition with the novel small-molecule inhibitor BDP-9066 prevented radiation-driven increases in motility both in vitro and in a clinically relevant orthotopic xenograft model of GBM. Crucially, treatment with BDP-9066 in combination with RT significantly increased survival in this model and markedly reduced infiltration of the contralateral cerebral hemisphere.Significance: An effective new strategy for the treatment of glioblastoma uses a novel, anti-invasive chemotherapeutic to prevent infiltration of the normal brain by glioblastoma cells.Cancer Res; 78(22); 6509–22. ©2018 AACR.

Subconfluent migration assays in the presence of 100nM BDP-9066

Summary of the properties of BDP-9066

Subconfluent migration assay in the presence of DMSO

Antibodies used for western blotting

untreated GFP E2 cells in an ex vivo brain slice motility assay

Supplemental figure legends

Ex vivo migration assay in the presence of BDP-9066.

Supplementary video figure legend

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.Significance: 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. Cancer Res; 78(8); 2096–114. ©2018 AACR.

Subconfluent migration assays in the presence of 1000nM BDP-9066

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. Significance: 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. Cancer Res; 78(8); 2096–114. ©2018 AACR.

Glioblastoma (GBM) is an aggressive and incurable primary brain tumor that causes severe neurologic, cognitive, and psychologic symptoms. Symptoms are caused and exacerbated by the infiltrative properties of GBM cells, which enable them to pervade the healthy brain and disrupt normal function. Recent research has indicated that although radiotherapy (RT) remains the most effective component of multimodality therapy for patients with GBM, it can provoke a more infiltrative phenotype in GBM cells that survive treatment. Here, we demonstrate an essential role of the actin-myosin regulatory kinase myotonic dystrophy kinase-related CDC42-binding kinase (MRCK) in mediating the proinvasive effects of radiation. MRCK-mediated invasion occurred via downstream signaling to effector molecules MYPT1 and MLC2. MRCK was activated by clinically relevant doses per fraction of radiation, and this activation was concomitant with an increase in GBM cell motility and invasion. Furthermore, ablation of MRCK activity either by RNAi or by inhibition with the novel small-molecule inhibitor BDP-9066 prevented radiation-driven increases in motility both in vitro and in a clinically relevant orthotopic xenograft model of GBM. Crucially, treatment with BDP-9066 in combination with RT significantly increased survival in this model and markedly reduced infiltration of the contralateral cerebral hemisphere. Significance: An effective new strategy for the treatment of glioblastoma uses a novel, anti-invasive chemotherapeutic to prevent infiltration of the normal brain by glioblastoma cells.Cancer Res; 78(22); 6509–22. ©2018 AACR.

The actin and microtubule cytoskeletons are critically important for cancer cell proliferation, and drugs that target microtubules are widely-used cancer therapies. However, their utility is compromised by toxicities due to dose and exposure. To overcome these issues, we characterized how inhibition of the actin and microtubule cytoskeleton regulatory LIM kinases could be used in drug combinations to increase efficacy. A previously-described LIMK inhibitor (LIMKi) induced dose-dependent microtubule alterations that resulted in significant mitotic defects, and increased the cytotoxic potency of microtubule polymerization inhibitors. By combining LIMKi with 366 compounds from the GSK Published Kinase Inhibitor Set, effective combinations were identified with kinase inhibitors including EGFR, p38 and Raf. These findings encouraged a drug discovery effort that led to development of CRT0105446 and CRT0105950, which potently block LIMK1 and LIMK2 activity in vitro, and inhibit cofilin phosphorylation and increase αTubulin acetylation in cells. CRT0105446 and CRT0105950 were screened against 656 cancer cell lines, and rhabdomyosarcoma, neuroblastoma and kidney cancer cells were identified as significantly sensitive to both LIMK inhibitors. These large-scale screens have identified effective LIMK inhibitor drug combinations and sensitive cancer types. In addition, the LIMK inhibitory compounds CRT0105446 and CRT0105950 will enable further development of LIMK-targeted cancer therapy.

As part of a program to develop a small molecule inhibitor of LIMK, a series of aminothiazole inhibitors were discovered by high throughput screening. Scaffold hopping and subsequent SAR directed development led to a series of low nanomolar inhibitors of LIMK1 and LIMK2 that also inhibited the direct biomarker p-cofilin in cells and inhibited the invasion of MDA MB-231-luc cells in a matrigel inverse invasion assay.

Myotonic dystrophy kinase-related CDC42-binding kinases MRCKα and MRCKβ regulate actin-myosin contractility and are members of the AGC family of serine/threonine kinases. MRCKs are closely related to the Rho-regulated ROCK kinases, which is reflected in their shared abilities to phosphorylate a similar set of substrates, including myosin II light chain proteins (MLC) and myosin phosphatase target subunit 1 (MYPT1). However, MRCK and ROCK may phosphorylate these substrates at different subcellular localisations leading to diverging effects on cell invasion and migration. Glioblastoma (GBM) is an aggressive and incurable primary brain tumor. Patients are treated with surgery, radiotherapy and chemotherapy but prognosis remains poor, with a median survival of 15 months. Complete surgical resection of GBM is difficult because these tumours are frequently invasive. Recent research has indicated that, while radiotherapy extends life expectancy of patients, it can provoke a more infiltrative phenotype in those GBM cells that survive treatment. We have developed potent and selective small molecule MRCK inhibitors exemplified by BDP-9066, which inhibits pMLC in a selective cellular assay with an EC50 28.3 nM, whilst displaying high selectivity over ROCK1 where the EC50 10.6 µM. This compound, and others in the series, have been used in numerous cellular assay systems to demonstrate that MRCK inhibition suppresses the invasion and migration of cancer cells including, breast, skin SCC and glioblastoma. We have used BDP-9066 to demonstrate that MRCK inhibition is effective at perturbing radiation-induced migration of GBM cells in culture at concentrations that block phosphorylation of MLC. Using a clinically relevant intracranial mouse model that recapitulates key histological features of the disease, we have shown that BDP-9066 inhibits the invasion of G7 GBM cells into the contralateral hemisphere of the brain. These experiments highlight the potential utility of MRCK inhibition in the treatment of GBM in patients. Citation Format: Heather J. McKinnon, Joanna Birch, Laura McDonald, Mairi Sime, Daniel Croft, Diane Crighton, Martin Drysdale, Lesley Gilmore, Christopher Gray, Jennifer Konczal, Duncan McArthur, Patricia McConnell, Mokdad Mezna, Alexander Schuettelkopf, Karen Strathdee, Justin Bower, Michael F. Olson, Anthony J. Chalmers. A novel small molecule inhibitor of MRCK shows utility in blocking radiation induced invasion of glioblastoma cells in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1933.

Leading cells require LIMK for matrix degradation and invadopodia formation during collective cell migration., LIM kinases 1 and 2 (LIMK1/2) are centrally positioned regulators of actin cytoskeleton dynamics. Using siRNA-mediated knockdown or a novel small molecule inhibitor, we show LIMK is required for path generation by leading tumor cells and nontumor stromal cells during collective tumor cell invasion. LIMK inhibition lowers cofilin phosphorylation, F-actin levels, serum response factor transcriptional activity and collagen contraction, and reduces invasion in three-dimensional invasion assays. Although motility was unaffected, LIMK inhibition impairs matrix protein degradation and invadopodia formation associated with significantly faster recovery times in FRAP assays indicative of reduced F-actin stability. When LIMK is knocked down in MDA-MB-231 cells, they lose the ability to lead strands of collectively invading cells. Similarly, when LIMK activity is blocked in cancer-associated fibroblasts, they are unable to lead the collective invasion of squamous carcinoma cells in an organotypic skin model. These results show that LIMK is required for matrix remodeling activities for path generation by leading cells in collective invasion.

Transforming growth factor beta (TGFbeta)-stimulated clone-22 domain family member 1 (TSC-22D1) has previously been associated with enhanced apoptosis in several cell systems. In an attempt to identify novel factors that are involved in the control of cell death during mammary gland involution, we found that the mRNA for isoform 2 of TSC-22D1 was highly upregulated 24 h after forced weaning, when a dramatic increase in cell death occurred, closely following the expression of the known inducer of cell death during involution, TGFbeta3. This was paralleled by strongly increased TSC-22D1 isoform 2 protein levels in the luminal epithelium. In contrast, RNA and protein expression levels of the isoform 1 of TSC-22D1 did not change during development. Whereas isoform 2 induced cell death, isoform 1 suppressed TGFbeta-induced cell death and enhanced proliferation in mammary epithelial cell lines. Furthermore, four distinct forms of isoform 2 protein were detected in the mammary gland, of which only a 15-kDa form was associated with early involution. Our data describe novel opposing functions of the two mammalian TSC-22D1 isoforms in cell survival and proliferation, and establish the TSC-22D1 isoform 2 as a potential regulator of cell death during mammary gland involution.

Although the transcription factor NF-kappaB is most clearly linked to the inhibition of extrinsic apoptotic signals such as TNFalpha by upregulating known anti-apoptotic genes, NF-kappaB has also been proposed to be required for p53-induced apoptosis in transformed cells. However, the involvement of NF-kappaB in this process is poorly understood. Here we investigate this mechanism and show that in transformed MEFs lacking NF-kappaB (p65-null cells) genotoxin-induced cytochrome c release is compromised. To further address how NF-kappaB contributes to apoptosis, gene profiling by microarray analysis of MEFs was performed, revealing that NF-kappaB is required for expression of Noxa, a pro-apoptotic BH3-only protein that is induced by genotoxins and that triggers cytochrome c release. Moreover, we find that in the absence of NF-kappaB, genotoxin treatment cannot induce Noxa mRNA expression. Noxa expression had been shown to be regulated directly by genes of the p53 family, like p73 and p63, following genotoxin treatment. Here we show that p73 is activated after genotoxin treatment only in the presence of NF-kappaB and that p73 induces Noxa gene expression through the p53 element in the promoter. Together our data provides an explanation for how loss of NF-kappaB abrogates genotoxin-induced apoptosis.

SummaryInactivation of cell death is a major step in tumor development, and p53, a tumor suppressor frequently mutated in cancer, is a critical mediator of cell death. While a role for p53 in apoptosis is well established, direct links to other pathways controlling cell death are unknown. Here we describe DRAM (damage-regulated autophagy modulator), a p53 target gene encoding a lysosomal protein that induces macroautophagy, as an effector of p53-mediated death. We show that p53 induces autophagy in a DRAM-dependent manner and, while overexpression of DRAM alone causes minimal cell death, DRAM is essential for p53-mediated apoptosis. Moreover, analysis of DRAM in primary tumors revealed frequent decreased expression often accompanied by retention of wild-type p53. Collectively therefore, these studies not only report a stress-induced regulator of autophagy but also highlight the relationship of DRAM and autophagy to p53 function and damage-induced programmed cell death.

The tumour suppressor p53 has been shown to regulate RNA polymerase (pol) III transcription both in vitro and in vivo. We have characterized the regions of p53 that contribute to this effect. Repression of pol III transcription in vivo does not require residues 13-19 near the N-terminus of p53 that are highly conserved through evolution. However, amino acids 22 and 23 in the adjacent transactivation domain do contribute to the inhibition of pol III activity. Deletions within the central DNA-binding core domain (residues 102-292) of p53 can entirely abolish the repression function in these assays, despite the fact that pol III templates contain no recognized p53 binding site. Deletion or substitution within the C-terminal domain of p53 can also compromise its ability to repress pol III activity in vitro and in transfected cells. These observations reveal that repression of pol III transcription is a complex function involving multiple regions of p53 extending throughout much of the protein.

Background:\ud The myotonic dystrophy kinase-related CDC42-binding kinases MRCKα and MRCKβ regulate actin-myosin contractility and have been implicated in cancer metastasis. Along with the related ROCK1 and ROCK2 kinases, the MRCK proteins initiate signalling events that lead to contractile force generation which powers cancer cell motility and invasion. A potential strategy for cancer therapy is to reduce metastasis by blocking MRCK activity, either alone or in combination with ROCK inhibition. However, to date no potent small molecule inhibitors have been developed with selectivity towards MRCK.\ud \ud Results:\ud Screening a kinase-focused small molecule chemical library resulted in the identification of compounds with inhibitory activity towards MRCK. Medicinal chemistry combined with in vitro enzyme profiling led to the discovery of 4-chloro-1-(4-piperidyl)-N-[5-(2-pyridyl)-1H-pyrazol-4-yl]pyrazole-3-carboxamide (BDP00005290; abbreviated as BDP5290) as a potent MRCK inhibitor. X-ray crystallography of the MRCKβ kinase domain in complex with BDP5290 revealed how this ligand interacts with the nucleotide binding pocket. BDP5290 demonstrated marked selectivity for MRCKβ over ROCK1 or ROCK2 for inhibition of myosin II light chain (MLC) phosphorylation in cells. While BDP5290 was able to block MLC phosphorylation at both cytoplasmic actin stress fibres and peripheral cortical actin bundles, the ROCK selective inhibitor Y27632 primarily reduced MLC phosphorylation on stress fibres. BDP5290 was also more effective at reducing MDA-MB-231 breast cancer cell invasion through Matrigel than Y27632. Finally, the ability of human SCC12 squamous cell carcinoma cells to invade a three-dimensional collagen matrix was strongly inhibited by 2 μM BDP5290 but not the identical concentration of Y27632, despite equivalent inhibition of MLC phosphorylation.\ud \ud Conclusions:\ud BDP5290 is a potent MRCK inhibitor with activity in cells, resulting in reduced MLC phosphorylation, cell motility and tumour cell invasion. The discovery of this compound will enable further investigations into the biological activities of MRCK proteins and their contributions to cancer progression.

Tumor cells undergo changes in metabolism to meet their energetic and anabolic needs. It is conceivable that mechanisms exist to sense these changes and link them to pathways that eradicate cells primed for cancer development. We report that the tumor suppressor p53 activates a cell death priming mechanism that senses extracellular adenosine. Adenosine, the backbone of ATP, accumulates under conditions of cellular stress or altered metabolism. We show that its receptor, A2B, is upregulated by p53. A2B expression has little effect on cell viability, but ligand engagement activates a caspase- and Puma-dependent apoptotic response involving downregulation of antiapoptotic Bcl-2 proteins. Stimulation of A2B also significantly enhances cell death mediated by p53 and upon accumulation of endogenous adenosine following chemotherapeutic drug treatment and exposure to hypoxia. Since extracellular adenosine also accumulates within many solid tumors, this distinct p53 function links programmed cell death to both a cancer- and therapy-associated metabolic change.

A defining characteristic of cancer malignancy is invasion and metastasis. In some cancers (e.g. glioma), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality. It has been estimated that invasion and metastasis are responsible for 90% of cancer deaths. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment. A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer. We present a refined method for examining the contributions of candidate proteins to collective invasion. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

The central arbiter of cell fate in response to DNA damage is p53, which regulates the expression of genes involved in cell cycle arrest, survival and apoptosis. Although many responses initiated by DNA damage have been characterized, the role of actin cytoskeleton regulators is largely unknown. We now show that RhoC and LIM kinase 2 (LIMK2) are direct p53 target genes induced by genotoxic agents. Although RhoC and LIMK2 have well-established roles in actin cytoskeleton regulation, our results indicate that activation of LIMK2 also has a pro-survival function following DNA damage. LIMK inhibition by siRNA-mediated knockdown or selective pharmacological blockade sensitized cells to radio- or chemotherapy, such that treatments that were sub-lethal when administered singly resulted in cell death when combined with LIMK inhibition. Our findings suggest that combining LIMK inhibitors with genotoxic therapies could be more efficacious than single-agent administration, and highlight a novel connection between actin cytoskeleton regulators and DNA damage-induced cell survival mechanisms. Refereed/Peer-reviewed

MRCK alpha and MRCK beta (myotonic dystrophy kinase-related Cdc42-binding kinases) belong to a subfamily of Rho GTPase activated serine/threonine kinases within the AGC-family that regulate the actomyosin cytoskeleton. Reflecting their roles in myosin light chain (MLC) phosphorylation, MRCK alpha and MRCK beta influence cell shape and motility. We report further evidence for MRCK alpha and MRCK beta contributions to the invasion of cancer cells in 3-dimensional matrix invasion assays. In particular, our results indicate that the combined inhibition of MRCK alpha and MRCK beta together with inhibition of ROCK kinases results in significantly greater effects on reducing cancer cell invasion than blocking either MRCK or ROCK kinases alone. To probe the kinase ligand pocket, we screened 159 kinase inhibitors in an in vitro MRCK beta kinase assay and found 11 compounds that inhibited enzyme activity >80% at 3 mu M. Further analysis of three hits, Y-27632, Fasudil and TPCA-1, revealed low micromolar IC(50) values for MRCK alpha and MRCK beta. We also describe the crystal structure of MRCK beta in complex with inhibitors Fasudil and TPCA-1 bound to the active site of the kinase. These high-resolution structures reveal a highly conserved AGC kinase fold in a typical dimeric arrangement. The kinase domain is in an active conformation with a fully-ordered and correctly positioned alpha C helix and catalytic residues in a conformation competent for catalysis. Together, these results provide further validation for MRCK involvement in regulation of cancer cell invasion and present a valuable starting point for future structure-based drug discovery efforts.

The spread of tumor cells from primary sites often occurs as associated cell collectives. In this form of invasion, the contribution of cells leading the way may differ from those that follow. By implication, proteins that regulate the actin cytoskeleton, a major driver of cell motility, may have different roles depending on whether they are in leading or following cells. The LIM kinases 1 and 2 (LIMK) phosphorylate and inactivate the filamentous actin severing function of cofilin proteins. Using siRNA or pharmacological inhibitors, LIMK was found to be required in leading cells of collectively invading tumor cells, or in cancer-associated stromal fibroblasts, for effective extracellular matrix degradation that facilitates three-dimensional invasion. The decreased extracellular matrix degrading activities were associated with an inability to form the stable filamentous actin structures necessary to make matrix-degrading protrusive structures. However, LIMK was not required for cell motility or for path-following in associated collectives. These findings show that leading and following cells in collective invasion have different properties and indicate that targeting the activities in leading cells is sufficient to significantly inhibit tumor cell invasiveness.

The intricate regulation of cell survival and cell death is critical for the existence of both normal and transformed cells. Two factors central to these processes are p53 and NF kappa B, with both factors having ascribed roles in both promoting and repressing cell death. Not surprisingly, a number of studies have previously reported interplay between p53 and NF kappa B. The mechanistic basis behind these observations, however, is currently incomplete. We report here further insights into this interplay using a system where blockade of NF kappa B inhibits cell death from p53, but at the same time sensitizes cells to death by TNF alpha. We found in agreement with a recent report showing that NF kappa B is required for the efficient activation of the BH3-only protein Noxa by the p53 family member p73, that p53's ability to induce Noxa is also impeded by inhibition of NF kappa B. In contrast to the regulation by p73, however, blockade of NF kappa B downstream of p53 decreases Noxa protein levels without effects on Noxa mRNA. Our further analysis of the effects of NF kappa B inhibition on p53 target gene expression revealed that while most target genes analysed where unaffected by blockade of NF kappa B, the p53-mediated induction of the pro-apoptotic gene p53AIP1 was significantly dependent on NF kappa B. These studies therefore add further insight into the complex relationship of p53 and NF kappa B. In addition, since both Noxa and p53AIP1 have been shown to be important components of p53-mediated cell death responses, these findings may also indicate critical points where NF kappa B plays a pro-apoptotic role downstream of p53.

Mammary gland involution is characterised by a high degree of apoptosis. By identifying genes that are upregulated at this developmental stage, we aimed to discover key factors that are involved in the induction of mammary epithelial cell death and therefore present potential tumour suppressors for breast cancer. Among 96 genes recently identified as specifically upregulated early during involution were the transforming growth factor beta (TGFβ)-stimulated clone 22 homologue (TSC-22/TGFβ1-induced transcript 4) and TGFβ3 [1]. TGFβ3 has recently been shown to be necessary for induction of apoptosis during mammary gland involution, while TSC-22 overexpression can lead to cell death. We have therefore tested whether TSC-22 mRNA expression can be induced by TGFβ3 and whether it is involved in or necessary for TGFβ-induced apoptosis. We further show that TSC-22 can enhance TGFβ3-induced Smad response and epithelial cell death. In addition, overexpression of TSC-22 alone can induce a Smad response and apoptosis in mammary epithelial cell cultures, which is independent of p53. Further, we have performed tests to study the necessity for Smad proteins during TSC-22-induced apoptosis, and to establish the intracellular localisation of TSC-22. A pilot study on a small cohort of archival breast cancer cases, representing all stages of malignant progression, shows that TSC-22 protein was reduced or undetectable in 60% of breast carcinomas when compared with adjacent normal breast tissue, suggesting that TSC-22 could indeed be a potential novel tumour suppressor gene. We shall present data showing that methylation of the TSC-22 promoter is not involved in the reduction of TSC-22 protein in breast cancer.

It is clear that changes in autophagy and autophagy regulators occur during tumor development and that this can have profound effects in certain tumor settings. The fact that p53, a key tumor suppressor mutated in approximately 50% of human cancers, has now also been shown to induce autophagy, has placed autophagy center stage in the minds of those interested in the development and treatment of malignant disease. p53 is a transcription factor that responds to cellular stress and prevents the propagation of cells which may otherwise form a tumor. The recent discovery, therefore, of DRAM (damage-regulated autophagy modulator) as a new p53 target which modulates autophagy is a major step forward in understanding how p53 controls autophagy and how this relates to tumor suppression. DRAM is a lysosomal protein that is not only critical for the ability of p53 to induce autophagy, but also for p53's ability to induce programmed cell death--a facet of p53 considered central to its tumor-suppressive effects. The fact that DRAM is also inactivated in certain cancers underscores its importance and highlights the possibility that autophagy may have a more profound role in cancer than was first believed.

The tumor suppressor p53 is a potent inducer of tumor cell death, and strategies exist to exploit p53 for therapeutic gain. However, because about half of human cancers contain mutant p53, application of these strategies is restricted. p53 family members, in particular p73, are in many ways functional paralogs of p53, but are rarely mutated in cancer. Methods for specific activation of p73, however, remain to be elucidated. We describe here a minimal p53-derived apoptotic peptide that induced death in multiple cell types regardless of p53 status. While unable to activate gene expression directly, this peptide retained the capacity to bind iASPP - a common negative regulator of p53 family members. Concordantly, in p53-null cells, this peptide derepressed p73, causing p73-mediated gene activation and death. Moreover, systemic nanoparticle delivery of a transgene expressing this peptide caused tumor regression in vivo via p73. This study therefore heralds what we believe to be the first strategy to directly and selectively activate p73 therapeutically and may lead to the development of broadly applicable agents for the treatment of malignant disease.

The ability of a tumour cell to evade programmed cell death (apoptosis) is crucial in the development of cancer. The process of apoptosis is complex and involves the careful interplay of a host of signalling molecules. Cellular stresses, such as DNA-damage, can initiate apoptosis through multiple pathways, all of which eventually lead to eradication of damaged cells that may otherwise go on to form a tumour. Moreover, the relevance of this to combating cancer is very strong since several therapeutic agents used to treat malignant disease utilize the cells' apoptotic machinery. The purpose of this review is to provide an insight into what we know about how apoptosis is initiated by DNA-damaging agents, how pro- and anti-apoptotic signals converge in the execution of cell death, and how such mechanisms can be perturbed in cancer.

The tumor suppressor p53 is a transcription factor that controls cellular growth and proliferation. p53 targets include RNA polymerase (pol) III-dependent genes encoding untranslated RNAs such as tRNA and 5S rRNA. These genes are repressed through interaction of p53 with TFIIIB, a TATA-binding protein (TBP)-containing factor. Although many studies have shown that p53 binds to TBP, the significance of this interaction has remained elusive. Here we demonstrate that the TBP-p53 interaction is of functional importance for regulating RNA pol III-transcribed genes. Unlike RNA pol II-dependent promoter repression, overexpressing TBP can reverse inhibition of tRNA gene transcription by p53. p53 does not disrupt the direct interaction between the TFIIIB subunits TBP and Brf1, but prevents the association of Brf1 complexes with TFIIIC2 and RNA pol III. Using chromatin immunoprecipitation assays, we found that TFIIIB occupancy on tRNA genes markedly decreases following p53 induction, whereas binding of TFIIIC2 to these genes is unaffected. Together our results support the idea that p53 represses RNA pol III transcription through direct interactions with TBP, preventing promoter occupancy by TFIIIB.

RNA polymerase (pol) III synthesizes essential small RNAs, including tRNA and 5S rRNA. Wild-type p53 can repress pol III transcription both in vitro and in vivo. Many tumours carry substitutions in p53 which have selective effects on its functions. We identify tumour-derived mutations that compromise the ability of p53 to regulate pol III transcription. Furthermore, substitution R175H, the most common mutation in cancers, converts p53 from a repressor to an activator of pol III. Oncoproteins neutralize p53 in some tumours; we show that human papillomavirus E6 and cellular hdm2 can both release pol III from repression by p53. These data suggest that the restraining influence of p53 on pol III will be lost in many tumours. In addition to these features of sporadic cancers, some individuals inherit mutant forms of p53 and consequently suffer from Li-Fraumeni syndrome, showing genetic predisposition to certain malignancies. We find that pol III transcriptional activity is often highly elevated in primary fibroblasts from Li-Fraumeni patients, especially if the germline p53 mutation is followed by loss of the remaining allele. Our data suggest that p53 status can have a profound effect upon pol III transcription and hence on the biosynthetic capacity of cells.

The invasive potential of carcinomas greatly contributes to their ability to metastasize, a process which is estimated to cause 90% of all human cancer deaths. The LIM kinase (LIMK) family of Ser/Thr kinases sit at a hub of signaling pathways downstream of the Rho family of GTPases. Functionally, LIMK is directly involved in regulating the activity of cofilin, a family of proteins which modulate cell movement through reorganization of the actin cytoskeleton network. LIMK is up-regulated in a number of invasive cancer cell lines and metastatic breast and prostate tumors, whilst an increase in LIMK activity has been shown to cause increased cellular invasion in multiple model systems. We demonstrate that LIMK inhibition by siRNA reduces the invasive capacity of MDA-MB-231-Luc breast cancer cells in an in vitro matrigel invasion assay and reduces fibroblast lead collective invasion in a co-culture organotypic model. Potent, selective, small molecule LIMK inhibitors have been identified that inhibit LIMK in vitro with sub-nanomolar activity in biochemical assays and low micromolar activity in cells as measured by cofilin phosphorylation. Treatment of MDA-MB-231-Luc cells or tumor associated fibroblasts with these small molecule inhibitors decreased cell invasion in vitro with minimal cellular toxicity. Collectively, these data further validate LIMK as an anti-invasive therapeutic target in tumor cells and demonstrate the potential utility of LIMK inhibitors in metastatic disease. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C210.