Molecular Cell Biology Laboratory
In my laboratory we are studying Ras and Rho GTPase signalling in cell cycle progression, motility and apoptosis. The results of these efforts will help us determine how these signalling pathways contribute to tumour growth and metastasis.
Rho signalling pathways as a messenger of physical proliferative cues
Normal cells require both chemical signals, such as growth factors, and physical cues such as adhesion and sub-confluence to proliferate. Physical cues alone are generally insufficient for cell proliferation, instead they give cells the permission to proliferate in response to growth factors only when in the correct physical environment. The activity of Rho proteins is regulated by physical cues including integrin engagement, confluence and formation of adherens junctions, therefore, Rho proteins may act through their downstream effector pathways as the relays that convey information about the physical environment to the cell-cycle machinery. During tumourigenesis, elevated expression or hyper-activation of Rho proteins could mimic the physical cues normally required for proliferation, contributing to the unchecked growth of tumour cells independent of interactions with the extracellular matrix.
We have determined that RhoA co-operates with Ras to promote proliferation of murine fibroblasts and human colon carcinoma cells by suppressing the induction of growth-inhibitory levels of the cyclin dependent kinase inhibitor p21Waf1/Cip1 (p21) by the Ras/MAPK pathway. We also found that when the strength of the Ras signal is not sufficient to induce growth-inhibitory levels of p21, blocking Rho function leads to the accumulation of p21 protein resulting in growth arrest. We recently showed that in addition to Ras-induction of p21 transcription, Ras and the MAPK pathway elevate p21 levels through a cyclin D1 imposed block in proteasome-mediated degradation. The ability of Rho to suppress p21 is mediated, in part, through transcriptional repression. We are currently attempting to identify the signalling pathway regulated by Rho that regulates p21 transcription. Modulating p21 protein levels may be an effective cancer therapeutic strategy since small variations in p21 expression may switch its activity from being an assembly factor for cyclin/CDK complexes to an inhibitor of proliferation.
ROCK signalling in tumour cell invasion and metastasis
In addition to deregulated proliferation, tumour cells have altered morphological characteristics and, in the case of metastatic cells, acquire the ability to traverse tissue boundaries. Given the role of Rho proteins in the regulation of cytoskeletal structures and cell motility, and their elevated expression in some tumour cells, it has been suggested that the Rho pathway is involved in the morphological changes and metastatic behaviour of tumour cells. Recent research has shown that inhibition of the Rho effector ROCK, a Serine/Threonine protein kinase, reduces the invasiveness of cancer cells. In addition, ROCK I and ROCK II have been found to be highly expressed in some cancers relative to normal tissues. These studies are consistent with a contribution of ROCK to tumour cell invasiveness and metastasis.
In order to study ROCK signalling, we have utilized a conditional activation system for specifically activating ROCK II. Using this conditionally-activated version, we have determined that ROCK is sufficient to make tumour cells grown as subcutaneous xenografts increase tumour angiogenesis and locally invade surrounding stroma. We have characterized the downstream substrates of ROCK that contribute to morphological changes in vitro and, by association, which lead to in vivo tumour cell invasion. We are currently examining the downstream signalling pathways activated by ROCK that contribute to tumour invasiveness and angiogenesis.
ROCK activation and nuclear disintegration during apoptosis
Apoptosis is an evolutionarily conserved process in multicellular organisms that leads to the death and subsequent removal of redundant or excess cells. In the dying cell, a family of cysteine-proteases called caspases are responsible for the "execution" phase, which is characterised by morphological changes including cell contraction and dynamic membrane blebbing, one of the earliest described and most obvious aspects of apoptotic cell death. Contractile force generated by actin-myosin cytoskeletal structures has been implicated as the driving power behind cell contraction and the formation of membrane blebs and apoptotic bodies. Ultimately, the dead cell is packaged into membrane-clad apoptotic bodies that facilitate uptake by neighboring cells or by specialized phagocytic cells.
We found that activation of RhoA is not the general mechanism for apoptotic contraction and blebbing. Signalling downstream of Rho, however, was essential as inhibition of ROCK prevented apoptotic membrane blebbing in a range of cell types, while active ROCK I was sufficient for cell contraction and membrane blebbing. These data, therefore, are consistent with increased actin-myosin contractile force, driven by Rho-independent ROCK activity, being responsible for apoptotic membrane blebbing.
During apoptosis, ROCK I, but not ROCK II, is cleaved by caspase-3 at a conserved sequence that removes the autoinhibitory domain. The truncated kinase has an eight-fold higher specific activity in vitro relative to full-length protein in the absence of Rho. The enhanced kinase activity is sufficient to drive caspase-independent cell contraction and membrane blebbing, consistent with a direct effect of ROCK on the development of the apoptotic morphology.
We have determined that proteins involved in the regulation of actin-myosin contractility, including myosin light chain and LIMK, become phosphorylated during apoptosis in a ROCK-dependent manner and that activation of a ROCK I-ER fusion protein leads to phosphorylation of the same proteins ultimately leading to caspase-independent contraction and membrane blebbing. In addition, the myosin binding subunit (MYPT1) of the myosin light chain phosphatase is cleaved in apoptotic cells. We are currently mapping the caspase cleavage site of MYPT1 and determining the effects on phosphatase function. We are also examining how phosphorylation of ROCK I influences the specific activity of the kinase during apoptosis. One possibility is that the process of apoptosis has evolved such that myosin light chain phosphorylation, which is crucial for contractile force generation, is irrevocably elevated through the simultaneous caspase-mediated cleavage and activation of ROCK I and inactivation of MYPT1.
The nuclear envelope separates the cytoplasm from the nucleus and is composed of outer and inner membranes connected at nuclear pore complexes. Ultrastructural analysis has revealed that the nucleus is enveloped by an actin matrix with "knots" of filamentous actin associated with the nuclear envelope. Given the functions of Rho and ROCK in regulating actin cytoskeletal structures, this signalling pathway likely influences nuclear morphology. Within the inner nuclear membrane is the fibrous nuclear lamina that gives the nucleus form and structural rigidity. During apoptosis, the nuclear envelope is degraded, allowing fragmented DNA and nuclear proteins to be packaged into membrane-clad apoptotic bodies. Caspase activation during apoptosis leads to the cleavage of the nuclear A/C and B lamins. Although it has been proposed that disruption of the nuclear lamina architecture is responsible for the breakdown of the nuclear envelope, other factors likely play a significant role. One possibility is that actin cytoskeletal changes and contractile force generation induced by ROCK may contribute to the destruction of the nuclear envelope during apoptosis. Activation of ROCK I causes cells to contract, bleb and distort nuclear morphology without leading to nuclear envelope disruption, unless the strength of the nuclear lamina has been weakened by caspase-mediated degradation or by genetic deletion. We are currently attempting to identify how actin-myosin filaments are physically connected to the nucleus and the role of these connections in apoptotic nuclear disintegration.
Sprouty regulation of B-Raf signalling
Melanoma is the major cause of skin cancer mortality, this form of cancer is especially deadly in advanced stages when it is refractory to currently available treatments. The rate of fatality rises significantly if the cancer is not diagnosed at an early stage due to its ability to aggressively spread via lymphatic or blood vessels to various organs, including the liver, lungs or brain, as well as other sites. There are approximately 7,000 cases of melanoma diagnosed per year in the UK, however, the number of cases diagnosed increases yearly. In Scotland between 1979 and 1998, the age-standardised incidence rates tripled in males and doubled in females, likely because of the cumulative effects of increased exposure to ultraviolet radiation, due to factors such as increased foreign travel, the increasing popularity of sun beds and the depletion of the ozone layer, on an aging population.
Since melanoma can spread so aggressively and result in the development of metastatic tumours relatively resistant to treatment, the search has been on to identify genetic alterations that might lead to improved diagnostics and effective targeted therapeutics. It has been known for some time that the K-Ras and N-Ras GTPases are mutated in a significant percentage of melanoma tumours. In 2002, it was reported that a downstream Ras effector, the B-Raf Serine/Threonine kinase, is mutated in over 60% of melanoma. Subsequent studies have found B-Raf mutations in a significant proportion of papillary thyroid, colorectal and ovarian carcinomas. These findings emphasize the importance of the Ras and Raf signalling pathway in promoting tumourigenesis.
Mutations to Ras GTPases or B-Raf contribute to the initiation and progression of cancer by altering the normal regulation of this intracellular signalling pathway. As a consequence, Ras/B-Raf signalling contributes significantly to the development of numerous aspects of the malignant phenotype by promoting cell cycle progression, resistance to apoptotic stimuli, neo-vascularisation and tumour cell motility, invasiveness and metastasis.
Working downstream of Ras GTPases is a three component mitogen-activated protein kinase (MAPK) cascade, consisting of the Raf, MEK and ERK proteins. In melanoma, the MAPK cascade is frequently de-regulated resulting in constitutively elevated signalling, enhanced cell proliferation and resistance to apoptosis. The de-regulation can be classified into two types; direct activation via mutation to N-Ras K-Ras, or B-Raf, and indirect activation via reduced expression of negative regulatory proteins including the RASSF1A tumour suppressor and the Raf Kinase Inhibitor Protein. While the direct activating events of N-Ras, K-Ras and B-Raf mutation are mutually exclusive in all but a very few instances, the indirect activation events often occur in conjunction with N-Ras, K-Ras or B-Raf mutation.
We recently reported that Sprouty2 is highly-expressed in melanoma cell lines compared to normal melanocytes. The elevated expression levels in melanoma cell lines with mutated B-Raf and high levels of active ERK were surprising since Sprouty2 has been characterised as an inhibitor of MAPK signalling. Sprouty1 expression is reduced in human prostate cancer, while Sprouty2 expression is reduced in breast cancers, and as a result it has been proposed that Sprouty proteins normally function as tumour suppressors to negatively regulate tumour growth by inhibiting MAPK signalling. In addition, Sprouty2 influences ERK activity in melanoma cell lines expressing wild-type B-Raf, but has little or no influence on MAPK signalling in melanoma cells in which B-Raf has been mutated, likely because of reduced binding of Sprouty2 to mutant B-Raf. These findings suggest that the resistance of melanoma cells with mutant B-Raf to Sprouty-mediated negative feedback inhibition of MAPK signalling might be an important component of the oncogenic activity of mutant B-Raf and might play a contributory role in the initiation and/or progression of melanoma.
We propose to identify the sites phosphorylated on Sprouty2 and to examine their biological roles in modulating the effects of B-Raf on melanoma cell proliferation and metastatic potential. Antibody reagents developed during these studies may also be clinically useful in the diagnosis, classification and prognosis of melanoma. These studies will provide insight into the role of the Sprouty protein family in the initiation and/or progression of melanoma, with the potential benefit of optimizing B-Raf as a therapeutic target.
