Regulation of ROCK1 via Notch1 during breast cancer cell migration into dense matrices

Background The behaviour of tumour cells depends on factors such as genetics and the tumour microenvironment. The latter plays a crucial role in normal mammary gland development and also in breast cancer initiation and progression. Breast cancer tissues tend to be highly desmoplastic and dense matrix as a pre-existing condition poses one of the highest risk factors for cancer development. However, matrix influence on tumour cell gene expression and behaviour such as cell migration is not fully elucidated. Results We generated high-density (HD) matrices that mimicked tumour collagen content of 20 mg/cm3 that were ~14-fold stiffer than low-density (LD) matrix of 1 mg/cm3. Live-cell imaging showed breast cancer cells utilizing cytoplasmic streaming and cell body contractility for migration within HD matrix. Cell migration was blocked in the presence of both the ROCK inhibitor, Y-27632, and the MMP inhibitor, GM6001, but not by the drugs individually. This suggests roles for ROCK1 and MMP in cell migration are complicated by compensatory mechanisms. ROCK1 expression and protein activity, were significantly upregulated in HD matrix but these were blocked by treatment with a histone deacetylase (HDAC) inhibitor, MS-275. In HD matrix, the inhibition of ROCK1 by MS-275 was indirect and relied upon protein synthesis and Notch1. Inhibition of Notch1 using pooled siRNA or DAPT abrogated the inhibition of ROCK1 by MS-275. Conclusion Increased matrix density elevates ROCK1 activity, which aids in cell migration via cell contractility. The upregulation of ROCK1 is epigenetically regulated in an indirect manner involving the repression of Notch1. This is demonstrated from inhibition of HDACs by MS-275, which caused an upregulation of Notch1 levels leading to blockade of ROCK1 expression.

Dormant but migratory tumour cells in desmoplastic stroma of invasive ductal carcinomas

Mortality in breast cancer is linked to metastasis and recurrence yet there is no acceptable biological model for cancer relapse. We hypothesise that there might exist primary tumour cells capable of escaping surgery by migration and resisting radiotherapy and chemotherapy to cause cancer recurrence. We investigated this possibility in invasive ductal carcinoma (IDC) tissue and observed the presence of solitary primary tumour cells (SPCs) in the dense collagen stroma that encapsulates intratumoural cells (ICs). In IDC tissue sections, collagen was detected with either Masson's Trichrome or by second harmonics imaging. Cytokeratin-19 (CK-19) and vimentin (VIM) antibodies were, respectively, used to identify epithelial-derived tumour cells and to indicate epithelial to mesenchymal transition (EMT). Confocal/multiphoton microscopy showed that ICs from acini were mainly CK-19 +ve and were encapsulated by dense stromal collagen. Within the stroma, SPCs were detected by their staining for both CK-19 and VIM (confirming EMT). ICs and SPCs were subsequently isolated by laser capture microdissection followed by multiplex tandem-PCR studies. SPCs were found to be enriched for pro-migratory and anti-proliferative genes relative to ICs. In vitro experiments using collagen matrices at 20 mg/cm 3, similar in density to tumour matrices, demonstrated that SPC-like cells were highly migratory but dormant, phenotypes that recapitulated the genotypes of SPCs in clinical tissue. These data suggest that SPCs located at the breast cancer perimeter are invasive and dormant such that they may exceed surgical margins and resist local and adjuvant therapies. This study has important connotations for a role of SPCs in local recurrence.

The biology of breast tumor progression : acquisition of hormone independence and resistance to cytotoxic drugs

Many breast tumors appear to follow a predictable clinical pattern, being initially responsive to endocrine therapy and to cytotoxic chemotherapy but ultimately exhibiting a phenotype resistant to both modalities. Using the MCF-7 human breast cancer cell line as an example of an 'early' phenotype (estrogen and progesterone receptor positive, steroid responsive, low metastatic potential), we have isolated and characterized a series of hormone-independent but hormone-responsive variants (MIII and MCF7/LCC1). However, these variants remain responsive to both antiestrogens and cytotoxic drugs (methotrexate and colchicine). MIII and MCF7/LCCl cells appear to mimic some of the critical aspects of the early progression to a more aggressive phenotype. An examination of the phenotype of these cells suggests that some hormone-independent breast cancer cells are derived from hormone-dependent parental cells. The development of a hormone-independent phenotype can arise independently of acquisition of a cytotoxic drug resistant phenotype.

Mechanisms of tumour invasion and metastasis : emerging targets for therapy

The progression of a tumour from one of benign and delimited growth to one that is invasive and metastatic is the major cause of poor clinical outcome in cancer patients. The invasion and metastasis of tumours is a highly complex and multistep process that requires a tumour cell to modulate its ability to adhere, degrade the surrounding extracellular matrix, migrate, proliferate at a secondary site and stimulate angiogenesis. Knowledge of the process has greatly increased and this has resulted in the identification of a number of molecules that are fundamental to the process. The involvement of these molecules has been shown to relate not only to the survival and proliferation of the tumour cell but, also to the processes of tumour cell adhesion, migration, and the tumour cells ability to degrade and escape the primary site as well as play a role in angiogenesis. These molecules may provide important therapeutic targets that represent the ability to target specific steps in the process of invasion and metastasis and provide additional therapies. The review focuses on representative key targets in each of these processes and summarises the state of play in each case.

A linear-elastic model of anisotropic tumour growth

This paper examines the effect of anisotropic growth on the evolution of mechanical stresses in a linear-elastic model of a growing, avascular tumour. This represents an important improvement on previous linear-elastic models of tissue growth since it has been shown recently that spatially-varying isotropic growth of linear-elastic tissues does not afford the necessary stress-relaxation for a steady-state stress distribution upon reaching a nutrient-regulated equilibrium size. Time-dependent numerical solutions are developed using a Lax-Wendroff scheme, which show the evolution of the tissue stress distributions over a period of growth until a steady-state is reached. These results are compared with the steady-state solutions predicted by the model equations, and key parameters influencing these steady-state distributions are identified. Recommendations for further extensions and applications of this model are proposed.

B-Raf mutation : a key player in molecular biology of cancer

B-Raf is one of the more commonly mutated proto-oncogenes implicated in the development of cancers. In this review, we consider the mechanisms and clinical impacts of B-Raf mutations in cancer and discuss the implications for the patient in melanoma, thyroid cancer and colorectal cancer, where B-Raf mutations are particularly common.

Differential subcellular and extracellular localisations of proteins required for insulin-like growth factor- and extracellular matrix-induced signalling events in breast cancer progression

Background: Cancer metastasis is the main contributor to breast cancer fatalities as women with the metastatic disease have poorer survival outcomes than women with localised breast cancers. There is an urgent need to develop appropriate prognostic methods to stratify patients based on the propensities of their cancers to metastasise. The insulin-like growth factor (IGF)-I:IGF binding protein (IGFBP):vitronectin complexes have been shown to stimulate changes in gene expression favouring increased breast cancer cell survival and a migratory phenotype. We therefore investigated the prognostic potential of these IGF- and extracellular matrix (ECM) interaction-induced proteins in the early identification of breast cancers with a propensity to metastasise using patient-derived tissue microarrays. Methods: Semiquantitative immunohistochemistry analyses were performed to compare the extracellular and subcellular distribution of IGF- and ECM-induced signalling proteins among matched normal, primary cancer and metastatic cancer formalin-fixed paraffin-embedded breast tissue samples. Results: The IGF- and ECM-induced signalling proteins were differentially expressed between subcellular and extracellular localisations. Vitronectin and IGFBP-5 immunoreactivity was lower while β1 integrin immunoreactivity was higher in the stroma surrounding metastatic cancer tissues, as compared to normal breast and primary cancer stromal tissues. Similarly, immunoreactive stratifin was found to be increased in the stroma of primary as well as metastatic breast tissues. Immunoreactive fibronectin and β1 integrin was found to be highly expressed at the leading edge of tumours. Based on the immunoreactivity it was apparent that the cell signalling proteins AKT1 and ERK1/2 shuffled from the nucleus to the cytoplasm with tumour progression. Conclusion: This is the first in-depth, compartmentalised analysis of the distribution of IGF- and ECM-induced signalling proteins in metastatic breast cancers. This study has provided insights into the changing pattern of cellular localisation and expression of IGF- and ECM-induced signalling proteins in different stages of breast cancer. The differential distribution of these biomarkers could provide important prognostic and predictive indicators that may assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy.

Elevated YKL40 is associated with advanced prostate cancer (PCa) and positively regulates invasion and migration of PCa cells

Chitinase 3-like 1 (CHI3L1 or YKL40) is a secreted glycoprotein highly expressed in tumours from patients with advanced stage cancers, including prostate cancer (PCa). The exact function of YKL40 is poorly understood, but it has been shown to play an important role in promoting tumour angiogenesis and metastasis. The therapeutic value and biological function of YKL40 are unknown in PCa. The objective of this study was to examine the expression and function of YKL40 in PCa. Gene expression analysis demonstrated that YKL40 was highly expressed in metastatic PCa cells when compared with less invasive and normal prostate epithelial cell lines. In addition, the expression was primarily limited to androgen receptor-positive cell lines. Evaluation of YKL40 tissue expression in PCa patients showed a progressive increase in patients with aggressive disease when compared with those with less aggressive cancers and normal controls. Treatment of LNCaP and C4-2B cells with androgens increased YKL40 expression, whereas treatment with an anti-androgen agent decreased the gene expression of YKL40 in androgen-sensitive LNCaP cells. Furthermore, knockdown of YKL40 significantly decreased invasion and migration of PCa cells, whereas overexpression rendered them more invasive and migratory, which was commensurate with an enhancement in the anchorage-independent growth of cells. To our knowledge, this study characterises the role of YKL40 for the first time in PCa. Together, these results suggest that YKL40 plays an important role in PCa progression and thus inhibition of YKL40 may be a potential therapeutic strategy for the treatment of PCa.

Markers of genetic susceptibility in human environmental hygiene and toxicology : the role of selected CYP, NAT and GST genes

Inherited genetic traits co-determine the susceptibility of an individual to a toxic chemical. Special emphasis has been put on individual responses to environmental and industrial carcinogens, but other chronic diseases are of increasing interest. Polymorphisms of relevant xenobiotic metabolising enzymes may be used as toxicological susceptibility markers. A growing number of genes encoding enzymes involved in biotransformation of toxicants and in cellular defence against toxicant-induced damage to the cells has been identified and cloned, leading to increased knowledge of allelic variants of genes and genetic defects that may result in a differential susceptibility toward environmental toxicants. "Low penetrating" polymorphisms in metabolism genes tend to be much more common in the population than allelic variants of "high penetrating" cancer genes, and are therefore of considerable importance from a public health point of view. Positive associations between cancer and CYP1A1 alleles, in particular the *2C I462V allele, were found for tissues following the aerodigestive tract. Again, in most cases, the effect of the variant CYP1A1 allele becomes apparent or clearer in connection with the GSTM1 null allele. The CYP1B1 codon 432 polymorphism (CYP1B1*3) has been identified as a susceptibility factor in smoking-related head-and-neck squameous cell cancer. The impact of this polymorphic variant of CYP1B1 on cancer risk was also reflected by an association with the frequency of somatic mutations of the p53 gene. Combined genotype analysis of CYP1B1 and the glutathione transferases GSTM1 or GSTT1 has also pointed to interactive effects. Of particular interest for the industrial and environmental field is the isozyme CYP2E1. Several genotypes of this isozyme have been characterised which seem to be associated with different levels of expression of enzyme activity. The acetylator status for NAT2 can be determined by genotyping or by phenotyping. In the pathogenesis of human bladder cancer due to occupational exposure to "classical" aromatic amines (benzidine, 4-aminodiphenyl, 1-naphthylamine) acetylation by NAT2 is regarded as a detoxication step. Interestingly, the underlying European findings of a higher susceptibility of slow acetylators towards aromatic amines are in contrast to findings in Chinese workers occupationally exposed to aromatic amines which points to different mechanisms of susceptibility between European and Chinese populations. Regarding human bladder cancer, the hypothesis has been put forward that genetic polymorphism of GSTM1 might be linked with the occurrence of this tumour type. This supports the hypothesis that exposure to PAH might causally be involved in urothelial cancers. The human polymorphic GST catalysing conjugation of halomethanes, dihalomethanes, ethylene oxide and a number of other industrial compounds could be characterised as a class theta enzyme (GSTT1) by means of molecular biology. "Conjugator" and "non-conjugator" phenotypes are coincident with the presence and absence of the GSTT1 gene. There are wide variations in the frequencies of GSTT1 deletion (GSTT1 *0/0) among different ethnicities. Human phenotyping is facilitated by the GST activity towards methyl bromide or ethylene oxide in erythrocytes which is representative of the metabolic GSTT1 competence of the entire organism. Inter-individual variations in xenobiotic metabolism capacities may be due to polymorphisms of the genes coding for the enzymes themselves or of the genes coding for the receptors or transcription factors which regulate the expression of the enzymes. Also, polymorphisms in several regions of genes may cause altered ligand affinity, transactivation activity or expression levels of the receptor subsequently influencing the expression of the downstream target genes. Studies of individual susceptibility to toxicants and gene-environment interaction are now emerging as an important component of molecular epidemiology.

Elevated level of inhibin-α subunit is pro-tumourigenic and pro-metastatic and associated with extracapsular spread in advanced prostate cancer

The biological function of inhibin-a subunit (INHa) in prostate cancer (PCa) is currently unclear. A recent study associated elevated levels of INHa in PCa patients with a higher risk of recurrence. This prompted us to use clinical specimens and functional studies to investigate the pro-tumourigenic and pro-metastatic function of INHa. We conducted a cross-sectional study to determine a link between INHa expression and a number of clinicopathological parameters including Gleason score, surgical margin, extracapsular spread, lymph node status and vascular endothelial growth factor receptor-3 expression, which are well-established prognostic factors of PCa. In addition, using two human PCa cell lines (LNCaP and PC3) representing androgen-dependent and -independent PCa respectively, we investigated the biological function of elevated levels of INHa in advanced cancer. Elevated expression of INHa in primary PCa tissues showed a higher risk of PCa patients being positive for clinicopathological parameters outlined above. Overexpressing INHa in LNCaP and PC3 cells demonstrated two different and cell-type-specific responses. INHa-positive LNCaP demonstrated reduced tumour growth whereas INHa-positive PC3 cells demonstrated increased tumour growth and metastasis through the process of lymphangiogenesis. This study is the first to demonstrate a pro-tumourigenic and pro-metastatic function for INHa associated with androgen-independent stage of metastatic prostate disease. Our results also suggest that INHa expression in the primary prostate tumour can be used as a predictive factor for prognosis of PCa.

Growth factors in induction of epithelial-mesenchymal transition and metastasis

Epithelial to mesenchymal transition (EMT) has gained widespread acceptance over recent years as a mechanism by which normally sessile epithelial tumour cells can move away from the primary tumour and metastasize. This review article examines the role of a number of growth factors in inducing EMT, and the reverse process mesenchymal to epithelial transition. Unique and common intracellular signalling pathways are highlighted. A comprehensive understanding of the regulation of EMT will be critical in manipulating this process to develop novel anti-metastasis therapies.

Pharmacokinetics and tumor disposition of PEGylated, methotrexate conjugated poly-l-lysine dendrimers

Dendrimers have potential for delivering chemotherapeutic drugs to solid tumours via the enhanced permeation and retention (EPR) effect. The impact of conjugation of hydrophobic anticancer drugs to hydrophilic PEGylated dendrimer surfaces, however, has not been fully investigated. The current study has therefore characterised the effect on dendrimer disposition of conjugating α-carboxyl protected methotrexate (MTX) to a series of PEGylated 3H-labelled poly-L-lysine dendrimers ranging in size from generation 3 (G3) to 5 (G5) in rats. Dendrimers contained 50% surface PEG and 50% surface MTX. Conjugation of MTX generally increased plasma clearance when compared to conjugation with PEG alone. Conversely, increasing generation reduced clearance, increased metabolic stability and reduced renal elimination of the administered radiolabel. For constructs with molecular weights >20 kDa increasing the molecular weight of conjugated PEG also reduced clearance and enhanced metabolic stability but had only a minimal effect on renal elimination. Tissue distribution studies revealed retention of MTX conjugated smaller (G3-G4) PEG570 dendrimers (or their metabolic products) in the kidneys. In contrast, the larger G5 dendrimer was concentrated more in the liver and spleen. The G5 PEG1100 dendrimer was also shown to accumulate in solid Walker 256 and HT1080 tumours and comparative disposition data in both rats (1 to 2% dose/g in tumour) and mice (11% dose/g in tumour) are presented. The results of this study further illustrate the potential utility of biodegradable PEGylated poly-L-lysine dendrimers as long circulating vectors for the delivery and tumour-targeting of hydrophobic drugs.

Protection by methylproamine of irradiated human keratinocytes correlates with reduction of DNA damage

Purpose: The therapeutic ratio for ionising radiation treatment of tumour is a trade-off between normal tissue side-effects and tumour control. Application of a radioprotector to normal tissue can reduce side-effects. Here we study the effects of a new radioprotector on the cellular response to radiation. Methylproamine is a DNA-binding radioprotector which, on the basis of published pulse radiolysis studies, acts by repair of transient radiation-induced oxidative species on DNA. To substantiate this hypothesis, we studied protection by methylproamine at both clonogenic survival and radiation-induced DNA damage, assessed by γH2AX (histone 2AX phosphorylation at serine 139) focus formation endpoints. Materials and methods: The human keratinocyte cell line FEP1811 was used to study clonogenic survival and yield of γH2AX foci following irradiation (137Cs γ-rays) of cells exposed to various concentrations of methylproamine. Uptake of methylproamine into cell nuclei was measured in parallel. Results: The extent of radioprotection at the clonogenic survival endpoint increased with methylproamine concentration up to a maximum dose modification factor (DMF) of 2.0 at 10 μM. At least 0.1 fmole/nucleus of methylproamine is required to achieve a substantial level of radioprotection (DMF of 1.3) with maximum protection (DMF of 2.0) achieved at 0.23 fmole/nucleus. The γH2AX focus yield per cell nucleus 45 min after irradiation decreased with drug concentration with a DMF of 2.5 at 10 μM. Conclusions: These results are consistent with the hypothesis that radioprotection by methylproamine is mediated by attenuation of the extent of initial DNA damage.