Acquired resistance to EGFR tyrosine kinase inhibitors alters the metabolism of human head and neck squamous carcinoma cells and xenograft tumours.

BACKGROUND: Acquired resistance to molecularly targeted therapeutics is a key challenge in personalised cancer medicine, highlighting the need for identifying the underlying mechanisms and early biomarkers of relapse, in order to guide subsequent patient management. METHODS: Here we use human head and neck squamous cell carcinoma (HNSCC) models and nuclear magnetic resonance (NMR) spectroscopy to assess the metabolic changes that follow acquired resistance to EGFR tyrosine kinase inhibitors (TKIs), and which could serve as potential metabolic biomarkers of drug resistance. RESULTS: Comparison of NMR metabolite profiles obtained from control (CAL(S)) and EGFR TKI-resistant (CAL(R)) cells grown as 2D monolayers, 3D spheroids or xenograft tumours in athymic mice revealed a number of differences between the sensitive and drug-resistant models. In particular, we observed elevated levels of glycerophosphocholine (GPC) in CAL(R) relative to CAL(S) monolayers, spheroids and tumours, independent of the growth rate or environment. In addition, there was an increase in alanine, aspartate and creatine+phosphocreatine in resistant spheroids and xenografts, and increased levels of lactate, branched-chain amino acids and a fall in phosphoethanolamine only in xenografts. The xenograft lactate build-up was associated with an increased expression of the glucose transporter GLUT-1, whereas the rise in GPC was attributed to inhibition of GPC phosphodiesterase. Reduced glycerophosphocholine (GPC) and phosphocholine were observed in a second HNSCC model probably indicative of a different drug resistance mechanism. CONCLUSIONS: Our studies reveal metabolic signatures associated not only with acquired EGFR TKI resistance but also growth pattern, microenvironment and contributing mechanisms in HNSCC models. These findings warrant further investigation as metabolic biomarkers of disease relapse in the clinic.

Selective FLT3 inhibition of FLT3-ITD+ acute myeloid leukaemia resulting in secondary D835Y mutation: a model for emerging clinical resistance patterns.

Acquired resistance to selective FLT3 inhibitors is an emerging clinical problem in the treatment of FLT3-ITD(+) acute myeloid leukaemia (AML). The paucity of valid pre-clinical models has restricted investigations to determine the mechanism of acquired therapeutic resistance, thereby limiting the development of effective treatments. We generated selective FLT3 inhibitor-resistant cells by treating the FLT3-ITD(+) human AML cell line MOLM-13 in vitro with the FLT3-selective inhibitor MLN518, and validated the resistant phenotype in vivo and in vitro. The resistant cells, MOLM-13-RES, harboured a new D835Y tyrosine kinase domain (TKD) mutation on the FLT3-ITD(+) allele. Acquired TKD mutations, including D835Y, have recently been identified in FLT3-ITD(+) patients relapsing after treatment with the novel FLT3 inhibitor, AC220. Consistent with this clinical pattern of resistance, MOLM-13-RES cells displayed high relative resistance to AC220 and Sorafenib. Furthermore, treatment of MOLM-13-RES cells with AC220 lead to loss of the FLT3 wild-type allele and the duplication of the FLT3-ITD-D835Y allele. Our FLT3-Aurora kinase inhibitor, CCT137690, successfully inhibited growth of FLT3-ITD-D835Y cells in vitro and in vivo, suggesting that dual FLT3-Aurora inhibition may overcome selective FLT3 inhibitor resistance, in part due to inhibition of Aurora kinase, and may benefit patients with FLT3-mutated AML.

Isolated groin recurrence in vulval squamous cell cancer (VSCC). The importance of node count.

OBJECTIVE: To determine whether there is a node count which can define an adequate inguinofemoral lymphadenectomy (IFL) in primary VSCC. METHODS: A retrospective and prospective review of patients with node negative VSCC who had a full staging IFL. Detection of isolated groin recurrences (IGR) would allow groins with higher risk of groin recurrence to be identified. RESULTS: The median node count of 228 IFLs in 139 patients was eight (0-24). There were six IGR (4.3%). Increased rate of IGR was present in patients with increased age, tumour diameter and depth of invasion, lymphovascular space invasion, unilateral IFL, and moderate/poor tumour grade. In the 138 groins with node counts of eight or greater there were no IGRs compared to six in the patients with either undissected groins or groin node counts less than eight (p = 0.030) Interval to IGR was significantly shorter than other sites of recurrence. Both disease-specific and overall survival were significantly reduced in IGR. CONCLUSIONS: An inadequate IFL is a nodal count of less than eight per groin; both these groins and undissected groins are at increased risk of IGR and should have close surveillance.

Guidelines for the welfare and use of animals in cancer research.

Animal experiments remain essential to understand the fundamental mechanisms underpinning malignancy and to discover improved methods to prevent, diagnose and treat cancer. Excellent standards of animal care are fully consistent with the conduct of high quality cancer research. Here we provide updated guidelines on the welfare and use of animals in cancer research. All experiments should incorporate the 3Rs: replacement, reduction and refinement. Focusing on animal welfare, we present recommendations on all aspects of cancer research, including: study design, statistics and pilot studies; choice of tumour models (e.g., genetically engineered, orthotopic and metastatic); therapy (including drugs and radiation); imaging (covering techniques, anaesthesia and restraint); humane endpoints (including tumour burden and site); and publication of best practice.

Cross-platform Q-TOF validation of global exo-metabolomic analysis: application to human glioblastoma cells treated with the standard PI 3-Kinase inhibitor LY294002.

The reproducibility of a metabolomics method has been assessed to identify changes in tumour cell metabolites. Tissue culture media extracts were analyzed by reverse phase chromatography on a Waters Acquity T3 column with a 13 min 0.1% formic acid: acetonitrile gradient on Agilent and Waters LC-Q-TOF instruments. Features (m/z, RT) were extracted by MarkerLynx (Waters) and Molecular Feature Extractor (Agilent) in positive and negative ionization modes. The number of features were similar on both instruments and the reproducibility of ten replicates was <35% signal variability for approximately 50% and 40% of all ions detected in positive and negative ionization modes, respectively. External standards spiked to the matrix showed CVs <25% in peak areas within and between days. U87MG glioblastoma cells exposed to the PI 3-Kinase inhibitor LY294002 showed significant alterations of several confirmed features. These included glycerophosphocholine, already shown by NMR to be modulated by LY294002, highlighting the power of this technology for biomarker discovery.

Phospholipase C gamma 1 regulates the Rap GEF1-Rap1 signalling axis in the control of human prostate carcinoma cell adhesion.

Phospholipase Cgamma1 (PLCgamma1) is activated downstream of a variety of extracellular stimuli and has previously been implicated in the regulation of motility responses central to tumour cell invasion. In this study, we used a novel RNAi vector system to achieve conditional PLCgamma1 knockdown in PC3LN3 human prostate carcinoma cells for further evaluation of PLCgamma1 in tumour cell biology. Using this approach, we revealed a role for PLCgamma1 in the regulation of PC3LN3 cell adhesion that appears to be independent of its effects on tumour cell chemotactic migration and spreading in response to extracellular matrix. Subsequent microarray analysis of PLCgamma1-knockdown cells revealed Rap GEF1 mRNA to be decreased in response to PLCgamma1 loss. This translated into a decrease in Rap GEF1 protein levels and a significant loss of Rap1 activity in PLCgamma1-knockdown cells. Transient knockdown of Rap GEF1 caused a reduction in PC3LN3 adhesion while overexpression of Rap GEF1 rescued the PLCgamma1 knockdown-induced adhesion defect. These data highlight control of the Rap GEF1-Rap1 molecular switch as a specific requirement for PLCgamma1-mediated tumour cell adhesion.

Gefitinib (ZD1839, Iressa) as palliative treatment in recurrent or metastatic head and neck cancer.

To assess the level of activity and toxicity of gefitinib (ZD1839, Iressatrade mark) in a population of patients with locally recurrent and/or metastatic head and neck cancer. Patients were recruited into an expanded access programme through the multidisciplinary head and neck clinics at the Royal Marsden and St George's Hospitals. Patients were required to have received at least one course of standard systemic chemotherapy or radiation therapy, or be medically unfit for chemotherapy. Patients were commenced on single-agent gefitinib at a dose of 500 mg day(-1). Clinical, symptomatic and radiological response, time to progression (TTP), survival and toxicity were recorded. A total of 47 patients were enrolled (35 male and 12 female) with a median age of 62 years (range 18-93 years). The observed clinical response rate was 8% with a disease control rate (complete response, partial response, stable disease) of 36%. In all, 34% of patients experienced an improvement in their symptoms. The median TTP and survival were 2.6 and 4.3 months, respectively. Acneiform folliculitis was the most frequent toxicity observed (76%) but the majority of cases were grade 1 or 2. Only four patients experienced grade 3 toxicity of any type (all cases of folliculitis). Gefitinib was well tolerated and yielded symptomatic improvement in one-third of patients. However, this agent appeared to possess limited antitumour activity in this group of patients with head and neck cancer in whom the objective response rate, median TTP and survival were all lower than has been reported in a previous study.

C-erbB receptors in squamous cell carcinomas of the head and neck: clinical significance and correlation with matrix metalloproteinases and vascular endothelial growth factors.

We studied the profile of four c-erbB receptors in head and neck squamous cell carcinomas (HNSCC) and to determine whether their expression was associated with clinicopathological features and key molecules involved in angiogenesis and metastasis. We also assessed the impact of expression on survival. This study included 54 cases of primary HNSCC, of which 27 cases showed lymph node metastasis. The expression of c-erbB receptors, matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) family members was analysed in the same tissue homogenates by semi-quantitative RT-PCR. HNSCC frequently co-expressed multiple c-erbB receptors and showed significant correlations amongst their levels. High expression of epidermal growth factor receptor (EGFR), c-erbB-2 or c-erbB-3 was associated with an infiltrating mode of invasion, nodal metastases and advanced pathological stages. EGFR and c-erbB-2 levels were strongly correlated (P=0.0004-0.029) with the expression of MMP-2, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, VEGF-A and VEGF-C whereas the levels of c-erbB-3 and B-4 showed a weaker correlation (P=0.049-0.01) with some MMPs and VEGF-C. Only nodal metastasis and EGFR levels were significantly associated with poor outcome in uni- and multi-variate analysis. We conclude that co-operative signalling of all four c-erbB receptors may play a significant role in the pathogenesis of HNSCC. Amongst these, EGFR appears to be the dominant component controlling the invasive and angio-/lymphangiogenic phenotype in HNSCC via upregulation of multiple MMPs and VEGFs.

Plasma basic fibroblast growth factor levels in colorectal cancer: a clinically useful assay?

Angiogenic cytokines in the plasma and serum of cancer patients may serve as 'surrogate' markers of tumour neoangiogenesis. Serum VEGF correlates with disease stage in colorectal cancer (CRC), but the role of bFGF in CRC is uncertain. This study aimed to assess plasma bFGF levels in CRC patients before treatment, during chemoradiotherapy and at one-year follow-up. Plasma samples were taken from 124 CRC patients, 26 polyp patients and 55 controls, and bFGF levels were measured by ELISA. 19 patients underwent pre-operative chemoradiotherapy. One-year follow-up samples were available from 48 disease-free patients and 18 patients with progressive disease. There were no detectable differences between plasma bFGF levels in polyp, Dukes' A or B patients (4.55, 5.77, 4.25 pg/ml, respectively), but there was a significant increase in metastatic CRC patients [Dukes' C and D (7.42 and 6.6 pg/ml; P = 0.004 and 0.048, respectively)], relative to median control levels of 4.14 pg/ml. At follow-up, there was a significant fall in plasma bFGF levels in disease-free patients (pre-op 6.09 and follow-up 3.45 pg/ml, P = 0.0004), but a non-significant rise in 18 patients with progressive disease (pre-treatment 5.90 and follow-up 9.99 pg/ml, P = 0.33). Pre-treatment plasma bFGF in patients receiving chemo-radiotherapy was similar in those with responsive and non-responsive tumours. There were no detectable changes in plasma bFGF through the adenoma-carcinoma sequence or patient groups with non-metastatic cancers. Elevated plasma bFGF was, however, associated with metastatic spread. The significant fall in bFGF in disease-free patients following therapy suggests that bFGF may be useful in clinical practice.

Non-invasive methods of assessing angiogenesis and their value in predicting response to treatment in colorectal cancer.

BACKGROUND: Tumour neoangiogenesis can be assessed non-invasively by measuring angiogenic cytokine concentrations in peripheral circulation and by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). The aim of this study was to assess whether these methods can predict and monitor response to treatment in patients with rectal cancer treated with preoperative chemoradiotherapy. METHODS: Serum and plasma vascular endothelial growth factor levels were measured in 31 patients with T3/T4 rectal cancers before quantitating tumour permeability (ln Ktrans) by DCE-MRI. Sixteen patients receiving preoperative chemoradiotherapy had serial vascular endothelial growth factor (VEGF) and DCE-MRI measurements. Response to treatment was assessed using World Health Organization criteria. RESULTS: Serum VEGF and ln Ktrans correlated before treatment (r = 0.48, P = 0.01). Responsive tumours (n = 8) had higher pretreatment permeability values than non-responsive tumours (n = 8) (mean ln Ktrans - 0.46 and - 0.72 respectively; P = 0.03). Compared with pretreatment values, responsive tumours showed a marked reduction in permeability at the end of treatment (mean ln Ktrans - 0.46 and - 0.86 respectively; P = 0.04). Pretreatment serum VEGF levels were not statistically different between the two groups. CONCLUSION: Rectal tumours with higher permeability at presentation appear to respond better to chemoradiotherapy than those of lower permeability. This may allow preselection of appropriate tumours for these regimens, with patients with low-permeability tumours being considered for alternative therapies.

VEGF-A, VEGF-C, and VEGF-D in colorectal cancer progression.

We aimed to assess the relationship of the angiogenic cytokines VEGF-A, VEGF-C, and VEGF-D and their receptors VEGFR-2 and VEGFR-3 in the adenoma-carcinoma sequence and in metastatic spread of colorectal cancer (CRC). mRNA expression levels were measured using semi-quantitative reverse transcription polymerase chain reaction in 70 CRC (35 with paired mucosae) and 20 adenomatous polyps. Immunohistochemistry and ELISA assessed protein expression. VEGF-D mRNA expression was significantly lower in both polyps and CRCs compared with normal mucosa (P=.0002 and.002, respectively), whereas VEGF-A and VEGF-C were significantly raised in CRCs (P=.006 and.004, respectively), but not polyps (P=.22 and P=.5, respectively). Receptor expression was similar in tumor tissue and normal mucosae. Tumors with lymph node metastases had significantly higher levels of VEGF-A compared with non-metastatic tumors (P=.043). There was no association between VEGF-C or VEGF-D and lymphatic spread. The decrease in VEGF-D occurring in polyps and carcinomas may allow the higher levels of VEGF-A and VEGF-C to bind more readily to the VEGF receptors, and produce the angiogenic switch required for tumor growth. Increased expression of VEGF-A within CRCs was associated with lymphatic metastases, and therefore, this member of the VEGF family may be the most important in determining metastatic spread.

Expression of matrix metalloproteinases and their inhibitors correlates with invasion and metastasis in squamous cell carcinoma of the head and neck.

BACKGROUND: Matrix metalloproteinases (MMPs) have been implicated in the invasion and metastasis of head and neck squamous cell carcinoma (HNSCC). However, a detailed analysis of MMPs and tissue inhibitors of MMPs (TIMPs) in relation to the biological behavior of HNSCC has yet to be performed in clinical material. OBJECTIVES: To study a comprehensive profile of MMPs and their 2 main inhibitors in HNSCC tissue samples and to correlate the patterns of expression with clinicopathological characteristics, invasion, and metastasis. DESIGN: This study included 54 consecutive patients with primary HNSCC, 27 of which showed lymph node metastasis. Expression of MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, MMP-14, TIMP-1, and TIMP-2 was simultaneously analyzed in tissue homogenates using semiquantitative reverse transcription-polymerase chain reaction assay. Where feasible, levels of protein and enzyme activity were confirmed by Western blot, enzyme-linked immunosorbent assay, and substrate zymography. Conventional clinicopathological features, including mode of tumor invasion, were also examined. RESULTS: Significantly higher MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, and TIMP-1 levels were found in tumors vs specimens of matched normal mucosa. No difference in the distribution of MMPs and TIMPs in relation to age, sex, tumor site, or histological grade was observed. A significant correlation was demonstrated between levels of MMP-1, MMP-9, and TIMP-1 and advanced T stage and between MMP-9 expression and an infiltrative pattern of growth. Enhanced expression of MMP-9 was strongly correlated (P<.001) and levels of MMP-2, MMP-7, and MMP-11 were weakly correlated (P =.03-.05) with lymph node involvement. CONCLUSIONS: Overexpression of multiple MMPs and TIMPs is characteristic of HNSCC, and analysis of specific MMPs, MMP-9 in particular, might be useful for evaluating the malignant potential in individual HNSCC.

Expression of vascular endothelial growth factor family members in head and neck squamous cell carcinoma correlates with lymph node metastasis.

BACKGROUND: The expression of vascular endothelial growth factor (VEGF)-A isoforms (121, 165, 189, 206), VEGF-B, VEGF-C and VEGF-D in both experimental and clinical models of head and neck squamous cell carcinoma (HNSCC) was determined and correlated with conventional clinicopathologic parameters, with particular reference to cervical nodal metastasis. METHODS: The mRNA expression of VEGFs in 14 HNSCC cell lines was compared with 4 normal keratinocyte cultures and 10 fibroblast cultures using a semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay. Protein levels were determined by Western blotting and enzyme-linked immunosorbent assay (ELISA). The authors then examined the expression of VEGFs in tissues from 54 patients including histologically normal epithelium (n = 32), early invasive squamous cell carcinomas (SCCs) (n = 23), advanced primary SCCs (n = 31), and lymph node metastases (n = 27). RESULTS: Increased levels of VEGF-A (all four isoforms) and VEGF-C were found in tumor cell lines compared with normal cells, whereas no differences in VEGF-B levels were found. VEGF-D expression, however, was lower in HNSCC cells. Studies in clinical samples showed highly significant increases in mRNA expression of all four isoforms of VEGF-A and VEGF-C in tumors versus normal epithelium. In contrast, the levels of VEGF-D were significantly decreased in tumors, and VEGF-B expression appeared similar in both normal and malignant tissues. Multivariate analysis demonstrated that an infiltrative mode of invasion and enhanced expression of VEGF-A (isoforms 121 and 165) and VEGF-C had predictive value for the presence of cervical nodal metastases. CONCLUSIONS: Up-regulation of VEGF-A (two isoforms) and VEGF-C and down-regulation of VEGF-D have been common features in HNSCC. Thus VEGF-A and VEGF-C appeared to play a vital role in the metastatic process of HNSCC.

Monoclonal antibodies targeting cancer: 'magic bullets' or just the trigger?

The first monoclonal antibodies (mAbs) approved for cancer therapy are now in Phase II and III trials, but the critical mechanism(s) determining efficacy and response in patients are still largely undefined. Both the direct antigen-binding (Fab) and constant (Fc) regions of mAbs can contribute to their biological activity. However, Clynes et al (Nat Med 2000, 6:443) recently suggested that the latter (at least in experimental models) might be the dominant component in vivo, triggering host responses to destroy cancer cells. Those workers showed that in mice lacking 'activation' Fc receptors (Fc(gamma)RI and Fc(gamma)RIII), anti-tumour effects of certain mAbs were significantly reduced. In contrast, mice deficient in the 'inhibitory' receptor Fc(gamma)RIIB responded with tumour growth inhibition and enhanced antibody-dependent cellular cytotoxicity (ADCC). These observations suggest that mAbs might be engineered for preferential binding to Fc(gamma)RIII to maximise therapeutic benefit. However, further work is needed to establish a definitive cause-effect relationship in experimental models that are more clinically relevant, to determine whether human Fc(gamma)R isoforms behave in a similar fashion, and to confirm that therapeutic mAbs and host cells can adequately access solid tumour deposits to mediate effective ADCC in situ. Finally, the 'cost-benefit' ratio of such modified macromolecules will need to be measured against mini-mAb constructs, antisense oligonucleotides, peptidomimetics and emerging drugs capable of inhibiting key tumour cell signalling pathways.

Basic principles for the study of metastasis using animal models.

Metastasis is the most devastating aspect of cancer, and the major reason for treatment failure. It is perhaps surprising, therefore, that it is only relatively recently that a wide variety of clinically relevant metastasis models have become generally available. For more than 20 years, the mainstays of cancer research were a handful of transplanted rodent tumors. A few of these (most notably B16F10 and Lewis lung carcinoma) were used as metastasis models, generally by injecting the cells intravenously to give lung colonies. The cancer research community and pharmaceutical industry could be criticized for coming up with few if any new drugs effective against solid tumor metastases. Yet is this surprising when for many years the "NCI screen" consisted of mouse ascites tumors? These are localized, "liquid" tumors, where drug access is direct and blood supply irrelevant, a poor model for solid deposits in a multiplicity of body compartments with varying vascular architecture and function. Such assays are more likely to produce drugs effective against leukemias (1).

The role of c-erbB-2/HER2/neu in breast cancer progression and metastasis.

Gene amplification and/or overexpression of the c-erbB-2/HER2/neu tyrosine kinase are linked with poor prognosis in breast cancer. This is manifest in shorter disease-free intervals, increased risk of metastasis, and resistance to many types of therapy. The molecular mechanisms and signaling circuitry underlying these phenomena are now being elucidated. c-erbB-2, although having no known soluble ligand, is transactivated by heterodimerization with other family members (EGFR, c-erbB-3, c-erbB-4). Receptor activation potentiates tumor cell motility, protease secretion and invasion, and also modulates cell cycle checkpoint function, DNA repair, and apoptotic responses. Since it is expressed at low levels in normal adult tissues, c-erbB-2 is an ideal target for therapy. There is reason for optimism that agents targeting c-erbB-2 signaling will have profound and selective effects in breast cancer, either as single agents or more likely in combination with other therapeutic agents, to enhance their potency.

Correlation of plasma and serum vascular endothelial growth factor levels with platelet count in colorectal cancer: clinical evidence of platelet scavenging?

Most studies measuring circulating vascular endothelial growth factor (VEGF) have sampled serum rather than plasma. There has been much debate whether the collection of sera (which causes the activation of platelets and VEGF release) is a true reflection of tumor angiogenic activity or whether platelets act as scavengers of VEGF. Addressing this issue, we measured serum and plasma VEGF, before and after colorectal resection, with reference to platelet counts. Serum and plasma samples were collected from 116 colorectal cancer (CRC) and 116 control patients. Ninety CRC and 32 benign resections were performed. Both plasma and serum VEGF were significantly higher in CRC patients (18.5 and 327 pg/ml, respectively) compared with controls (9.0 and 151.5 pg/ml, respectively; P < 0.0001). Paired serum and plasma VEGF measurements correlated in both CRC (r = 0.56) and control patients (r = 0.73; P < 0.0001). Serum and plasma VEGF levels correlated with platelet count in CRC patients (r = 0.58 and 0.44, respectively) but not in controls. Plasma and serum VEGF levels, and VEGF concentration per platelet, increased with advancing disease stage. The correlation of serum and plasma VEGF with platelet counts in CRC but not in benign disease may be attributable to the scavenging of VEGF from the tumor source by platelets, with plasma levels reflecting free circulating VEGF in equilibrium with platelet levels. VEGF levels in citrated plasma are low and lie close to the limits of ELISA sensitivity. We recommend that a standardized measurement of serum VEGF--normalized by the patient's platelet count to give a value of serum VEGF per platelet--be adopted.

Cell biology of lymphatic metastasis. The potential role of c-erbB oncogene signalling.

Lymphatic metastases are an important indicator of the malignancy of epithelial cancers. Empirical clinical observations associating specific genetic abnormalities with tumour progression, allied with basic laboratory investigations, are providing not only improved prognostic and diagnostic opportunities, but also a detailed understanding of the molecular machinery of metastasis. One such association--between the c-erbB oncogene family and metastasis--has proved particularly instructive. Functional links between over-expression (and occasionally mutational activation) of c-erbB-1 (EGFR) and c-erbB-2 and specific phenotypes of metastatic cells have been elucidated. Activated c-erbB oncogenes potentiate tumour cell adhesion to endothelial cells and upregulate VEGF, potentially facilitating angiogenesis and vascular invasion. In addition, cells over-expressing these oncogenes frequently show aberrant cell-cell and cell-matrix interactions, mediated by changes in integrin and cadherin function. Thirdly, both EGFR and c-erbB-2 signalling can significantly upregulate specific matrix metalloproteinases, key enzymes involved in angiogenesis and invasion. Finally, c-erbB receptors linked to the actin cytoskeleton and highly expressed on invadopodia, are thought to assist cell migration. Taken together, these observations suggest that such receptors can act as "master switches" in metastasis, whose activation co-ordinately controls events normally utilised in development, now subverted by the metastatic cell. As such, they represent ideal targets for therapeutic intervention.

Epidermal growth factor-like ligands differentially up-regulate matrix metalloproteinase 9 in head and neck squamous carcinoma cells.

Head and neck squamous cell carcinomas (HNSCCs) are characterized by a marked propensity for local invasion and dissemination to cervical lymph nodes, with distant metastases developing in 30-40% of cases. Overexpression of the epidermal growth factor receptor (EGFR/c-erbB-1) and/or its ligands and high levels of certain matrix metalloproteinases (MMPs) have been associated with poor prognosis. The aim of this study was to examine the effects of EGFR ligands on gelatinase expression and invasion in HNSCC cell lines. We tested epidermal growth factor (EGF), transforming growth factor alpha, betacellulin, heparin-binding EGF, and amphiregulin and measured expression of gelatinases MMP-9 and MMP-2 in an established squamous carcinoma cell line (Detroit-562) and in two cell lines newly derived from patients with head and neck cancers (SIHN-005A and SIHN-006). Incubation of the cell lines with EGF-like ligands up-regulated MMP-9 (but not MMP-2) expression as measured by semiquantitative reverse transcription-PCR in a dose-dependent manner, with the effects being most marked in cells with high EGFR levels and undetectable in cells with low levels. Maximum stimulation was obtained in a concentration range of 10-100 nM. In addition, we confirmed by zymography that gelatinolytic activity consistent with MMP-9 (Mr 92,000) was up-regulated in parallel with increases in gene expression. Betacellulin (which binds both to EGFR and c-erbB-4 receptors) consistently increased MMP-9 expression and activation to a significantly greater degree than the other four ligands when tested at equimolar concentrations. In parallel with MMP-9 up-regulation, all EGF-like ligands increased tumor cell invasion through Matrigel in in vitro Transwell assays. These activities were independent of ligand effects on cell proliferation. Antagonist (ICR62) or agonist (ICR9) anti-EGFR monoclonal antibodies, respectively, inhibited or potentiated MMP-9 activity and tumor cell invasion induced by all ligands. Furthermore, a monoclonal antibody that neutralizes MMP-9 activity (Abl) also inhibited ligand-induced invasion of HNSCC. We confirmed that tumor cell lines used in these studies (and a larger series not reported here) generally expressed multiple c-erbB receptors and ligands. These results indicate that autocrine or paracrine signaling through EGFR potentiates the invasive potential of HNSCC via the selective up-regulation and activation of MMP-9. Furthermore, ligands such as betacellulin (which is commonly expressed in HNSCC), which can bind to and activate other c-erbB receptors, may be especially potent in this regard.