Tumour cell metastasis involves cell adhesion and invasion, processes that depend on signal transduction, which can be influenced by the tumour microenvironment. N-6 polyunsaturated fatty acids, found both in the diet and in response to inflammatory responses, are important components of this microenvironment.
We used short hairpin RNA (shRNA) knockdown of TGF-β-activated kinase-1 (TAK1) in human tumour cells to examine its involvement in fatty acid-stimulated cell adhesion and invasion in vitro. An in vivo model of metastasis was developed in which cells, stably expressing firefly luciferase and either a control shRNA or a TAK1-specific shRNA, were injected into the mammary fat pads of mice fed diets, rich in n-6 polyunsaturated fatty acids. Tumour growth and spontaneous metastasis were monitored with in vivo and in situ imaging of bioluminescence.
Arachidonic acid activated TAK1 and downstream kinases in MDA-MB-435 breast cancer cells and led to increased adhesion and invasion. Knockdown of TAK1 blocked this activation and inhibited both cell adhesion and invasion in vitro. Tumour growth at the site of injection was not affected by TAK1 knockdown, but both the incidence and extent of metastasis to the lung were significantly reduced in mice injected with TAK1 knockdown cells compared with mice carrying control tumour cells.
These data demonstrate the importance of TAK1 signalling in tumour metastasis in vivo and suggest an opportunity for antimetastatic therapies.
adhesion; invasion; metastasis; fatty acid; TGF-β-activated kinase-1; breast cancer
PI3K/Akt (PKB) pathway has been shown in several cell types to be activated by ligands to cell surface integrins, leading to the metastasis of tumour cells. The signalling pathways involved in the metastatic spread of human scirrhous gastric carcinoma cells have not been defined.
The role of the PI3K/Akt pathway in an extensive peritoneal-seeding cell line, OCUM-2MD3 and a parental cell line, OCUM-2M, was investigated by assessing in vitro adhesion and spreading assay, and in vivo peritoneal metastatic model. We also examined the correlation of PI3K/Akt pathway with integrin signals by immunoprecipitations, using cells by transfection with mutant p85 (Δp85).
Adhesiveness and spreading of OCUM-2MD3 cells on collagen type IV was significantly decreased by PI3K inhibitors and expression of mutant p85, but not by inhibitors of protein kinase C (PKC) or extracellular signal-regulated kinase (ERK). Immunoprecipitation studies indicated that the PI3K/Akt pathway was associated with integrin signalling through Src and vinculin. In an in vivo experimental metastasis model, p85 inhibition reduced peritoneal metastasis of OCUM-2MD3 cells.
PI3K/Akt signalling may be required for integrin-dependent attachment and spreading of scirrhous gastric carcinoma cells, and would be translated into generating better strategies to optimise their use in cancer clinical trials.
PI3 kinase; gastric carcinoma; adhesion; spreading: metastasis; integrin signalling
The intake of dietary fatty acids is highly correlated with the risk of various cancers. Linoleic acid (LA) is the most abundant polyunsaturated fat in the western diet, but the mechanism(s) by fatty acids such as LA modulate cancer cells is unclear. In this study, we examined the role of LA in various steps in gastric cancer progression.
The difference in gene expression between LA-treated and untreated OCUM-2MD3 gastric carcinoma cells was examined by mRNA differential display. The involvement of candidate genes was examined by oligo- and plasmid-mediated RNA interference. Biological functions of several of these genes were examined using in vitro assays for invasion, angiogenesis, apoptosis, cell viability, and matrix digestion. Angiogenesis in vivo was measured by CD-31 immunohistochemistry and microvessel density scoring.
LA enhanced the plasminogen activator inhibitor 1 (PAI-1) mRNA and protein expression, which are controlled by PAI-1 mRNA-binding protein. LA-stimulated invasion depended on PAI-1. LA also enhanced angiogenesis by suppression of angiostatin, also through PAI-1. LA did not alter cell growth in culture, but increased dietary LA-enhanced tumour growth in an animal model.
Our findings suggest that dietary LA impacts multiple steps in cancer invasion and angiogenesis, and that reducing LA in the diet may help slow cancer progression.
gastric carcinoma; linoleic acid; plasminogen activator inhibitor 1; angiostatin; invasion
We reported that Notch-1, a potent breast oncogene, is activated in response to trastuzumab and contributes to trastuzumab resistance in vitro. We sought to determine the preclinical benefit of combining a Notch inhibitor (γ-secretase inhibitor (GSI)) and trastuzumab in both trastuzumab-sensitive and trastuzumab-resistant, ErbB-2-positive, BT474 breast tumours in vivo. We also studied if the combination therapy of lapatinib plus GSI can induce tumour regression of ErbB-2-positive breast cancer.
We generated orthotopic breast tumour xenografts from trastuzumab- or lapatinib-sensitive and trastuzumab-resistant BT474 cells. We investigated the antitumour activities of two distinct GSIs, LY 411 575 and MRK-003, in vivo.
Our findings showed that combining trastuzumab plus a GSI completely prevented (MRK-003 GSI) or significantly reduced (LY 411 575 GSI) breast tumour recurrence post-trastuzumab treatment in sensitive tumours. Moreover, combining lapatinib plus MRK-003 GSI showed significant reduction of tumour growth. Furthermore, a GSI partially reversed trastuzumab resistance in resistant tumours.
Our data suggest that a combined inhibition of Notch and ErbB-2 signalling pathways could decrease recurrence rates for ErbB-2-positive breast tumours and may be beneficial in the treatment of recurrent trastuzumab-resistant disease.
ErbB-2; trastuzumab; Notch-1; GSI; recurrence; resistance
Dietary (n-6)-polyunsaturated fatty acids influence cancer development, but the mechanisms have not been well characterised in gastric carcinoma.
We used two in vivo models to investigate the effects of these common dietary components on tumour metastasis. In a model of experimental metastasis, immunocompromised mice were fed diets containing linoleic acid (LA) at 2% (LLA), 8% (HLA) or 12% (VHLA) by weight and inoculated intraperitoneally (i.p.) with human gastric carcinoma cells (OCUM-2MD3). To model spontaneous metastasis, OCUM-2MD3 tumours were grafted onto the stomach walls of mice fed with the different diets. In in vitro assays, we investigated invasion and ERK phosphorylation of OCUM-2MD3 cells in the presence or absence of LA. Finally, we tested whether a cyclooxygenase (COX) inhibitor, indomethacin, could block peritoneal metastasis in vivo.
Both the HLA and VHLA groups showed increased incidence of tumour nodules (LA: 53% HLA: 89% VHLA: 100% P<0.03); the VHLA group also displayed increased numbers of tumour nodules and higher total volume relative to LLA group in experimental metastasis model. Both liver invasion (78%) and metastasis to the peritoneal cavity (67%) were more frequent in VHLA group compared with the LLA group (22% and 11%, respectively; P<0.03) in spontaneous metastasis model. We also found that the invasive ability of these cells is greatly enhanced when exposed to LA in vitro. Linoleic acid also increased invasion of other scirrhous gastric carcinoma cells, OCUM-12, NUGC3 and MKN-45. Linoleic acid effect on OCUM-2MD3 cells seems to be dependent on phosphorylation of ERK. The data suggest that invasion and phosphorylation of ERK were dependent on COX. Indomethacin decreased the number of tumours and total tumour volume in both LLA and VHLA groups. Finally, COX-1, which is known to be an important enzyme in the generation of bioactive metabolites from dietary fatty acids, appears to be responsible for the increased metastatic behaviour of OCUM-2MD3 cells in the mouse model.
Dietary LA stimulates invasion and peritoneal metastasis of gastric carcinoma cells through COX-catalysed metabolism and activation of ERK, steps that compose pathway potentially amenable to therapeutic intervention.
gastric carcinoma; dietary fatty acid; cyclooxygenase; metastasis; invasion
Among older mothers, preeclampsia in the first pregnancy was associated with a reduction in maternal breast cancer risk that was significantly more pronounced in women bearing male than female infants. Androgen concentrations in male, preeclamptic pregnancies were consistent with the hypothesis that elevated pregnancy androgens might mediate this apparent modifying effect of fetal gender.
preeclampsia; androgens; hormones; breast cancer; maternal; offspring gender
Adjuvant BEP (bleomycin, etoposide, cisplatin) is effective treatment for high-risk clinical stage I (HRCS1) non-seminomatous germ cell tumours (NSGCT), but the known toxicities of etoposide, and the expansion of the HR group to any patient with vascular invasion (50% of patients), led the Medical Research Council to pilot the BOP regimen. Patients received two courses of BOP 14 days apart: cisplatin 50 mg m−2 days 1 and 2, vincristine 1.4 mg m−2 (max. 2 mg) days 2 and 8, bleomycin 30 000 IU days 2 and 8. Primary outcome was relapse rate; quality of life, fertility, hearing and lung function were assessed pre- and post-treatment. In all, 100 patients were required. A total of 115 eligible patients were registered, all received two courses of chemotherapy. Median follow-up is 70 months; two relapses have occurred and the 5-year relapse-free rate is 98.3% (95% confidence interval (CI) 95.5%, 99.9%). As assessed by clinicians during treatment, complete (reversible) alopecia was present in 20% of patients; World Health Organization (WHO) grade 1/2 neurotoxicity was present in 41%/5% of patients during treatment and 22%/1% at 6 months. However, 12% of patients reported ‘quite a bit' or ‘very much' pain/numbness/tingling in hands/feet 2 years after chemotherapy. Mature follow-up confirms high efficacy for two courses of cisplatin-based adjuvant chemotherapy in HRCS1 NSGCT. Substituting vincristine for etoposide decreases alopecia, but gives a low incidence of significant neuropathy. There are no clearcut advantages to 2 × BOP over 2 × BEP, except for patients who wish to maximise the chance of avoiding significant alopecia.
adjuvant chemotherapy; stage I non-seminoma
malignant germ cell tumour; poor prognosis; chemotherapy; dose intense
testis; residual neoplasms; histology; statistical models; validity
The UK Medical Research Council conducted this trial of carboplatin chemotherapy in advanced seminoma to compare single agent carboplatin with a standard combination of etoposide with cisplatin. The use of single agent carboplatin was expected to be associated with reduced toxicity. A total of 130 patients with advanced seminoma were randomly assigned to treatment with either single agent carboplatin (C) at a dose of 400 mg/m2 to be corrected for glomerular filtration rate outside the range 81–120 ml min–1 and to be administered on day 1 of a 21 day cycle to a total of 4 cycles or to etoposide + platinum (EP). The trial was designed as an equivalence study aiming to exclude a reduction in the 3-year progression-free survival in patients allocated to carboplatin of between 10 and 15%, requiring initially a target accrual of 250 patients (90% power significance level 5% (one-sided)). The trial closed after 130 patients had been randomized following recommendation by an independent data monitoring committee. At a median follow-up time of 4.5 years, 81% of patients had been followed up for at least 3 years and 19 patients have died. The estimated PFS rate (95% Confidence Intervals (CI)) at 3 years was 71% (60–82%) in patients allocated C and 81% (71–90%) in those allocated EP; the 95% CI for the difference in 3 year PFS was – 6% to +19%. The hazard ratio of 0.64 (95% CI 0.32–1.28) favoured EP but the difference was not statistically significant (log rank chi-squared = 1.59 P = 0.21). The 3-year survival rate was 84% (75–92%) in those allocated C, and 89% (81–96%) in those allocated EP. The hazard ratio for survival was 0.85 with 95% CI, 0.35–2.10, log rank chi-squared = 0.12, P = 0.73. The trial has not demonstrated statistically significant differences in the major survival endpoints comparing single agent carboplatin with a combination of etoposide + cisplatin. This cannot be taken as an indication of equivalence since the limited size of this trial rendered it unable to exclude a 19% lower progression-free survival and survival in those treated with single agent carboplatin which would be important clinically. Standard initial chemotherapy for advanced seminoma should be based on cisplatin combinations and the role of carboplatin awaits the outcome of further studies. © 2000 Cancer Research Campaign http://www.bjcancer.com
seminoma; germ cell tumour; chemotherapy; cisplatin; carboplatin; randomized control trial
We attempted to identify factors associated with delay in presentation and assessment of women with breast symptoms who attended a London breast clinic. A total of 692 consecutive symptomatic referrals, aged 40–75 years, were studied. Patient delay, assessed prior to diagnosis, was defined as time elapsing between symptom discovery and first presentation to a medical provider. This was studied in relation to: reasons for delaying, beliefs and attitudes, socio-demographic and clinical variables, psychiatric morbidity and subsequent diagnosis. Thirty-five per cent of the cohort delayed presentation 4 weeks or more (median 13 days). The most common reason given was that they thought their symptom was not serious (odds ratio (OR) = 5.32, 95% confidence interval (CI) 3.6–8.0). Others thought their symptom would go away (OR = 3.73, 95% CI 2.2–6.4) or delayed because they were scared (OR = 4.61, 95% CI 2.1–10.0). Delay was associated with psychiatric morbidity but not age. Patients who turned out to have cancer tended to delay less (median 7 days) but not significantly. Median system delay – time between first medical consultation and first clinic visit – was 18 days. Patients who thought they had cancer and those so diagnosed were seen more promptly (median 14 days). Most factors, including socio-economic status and ethnicity were non-contributory. Beliefs about breast symptoms and their attribution are the most important factors determining when women present. Health education messages should aim to convince symptomatic women that their condition requires urgent evaluation, without engendering fear in them. © 2000 Cancer Research Campaign
patient delay; system delay; breast symptoms; breast cancer; reasons for delaying
Transitional cell carcinomas may arise at any site within the urinary tract and are a source of considerable morbidity and mortality. In particular, patients with metastatic disease have a poor prognosis, with less than 5% alive at 5 years. A multicentre randomized trial comparing methotrexate and vinblastine (MV) with cisplatin, methotrexate and vinblastine (CMV) in advanced or metastatic transitional cell carcinoma was conducted in the UK. From April 1991 to June 1995, 214 patients were entered by 16 centres, 108 randomized to CMV and 106 to MV. A total of 204 patients have died. The hazard ratio (relative risk of dying) was 0.68 (95% CI 0.51-0.90, P-value = 0.0065) in favour of CMV. This translates to an absolute improvement in 1-year survival of 13%, 16% in MV and 29% in CMV. The median survival for CMV and MV was 7 months and 4.5 months respectively. Two hundred and eight patients objectively progressed or died. The hazard ratio was 0.55 (95% CI 0.41-0.73, P-value = 0.0001) in favour of CMV. Two hundred and nine patients symptomatically progressed or died. The hazard ratio was 0.48 (95% CI 0.36-0.64, P-value = 0.0001) in favour of CMV. The most important pretreatment factors influencing overall survival were WHO performance status and extent of disease. These two factors were used to derive a prognostic index which could be used to categorize patients into three prognostic groups. We conclude that the addition of cisplatin to methotrexate and vinblastine should be considered in patients with transitional cell carcinoma, taking into account the increased toxicity.
The aim of the present retrospective study was to assess long-term survival after cisplatin-based chemotherapy in 398 patients with advanced urothelial transitional cell carcinoma (TCC) treated at seven international oncological units. Various combinations of cisplatin, methotrexate, vinblastine (or vincristine) and doxorubicin were used. The complete response rate according to the WHO criteria was 17%. Partial responses were obtained in 42% of the patients. The overall cancer-related 2 year and 5 year survival rates were 21% and 11% respectively. Based on multivariate analyses, a good prognosis group could be identified comprising patients with a good performance status with disease confined to lymph nodes (14%) or patients with T4b disease only. These patients had a 28% 5 year survival rate, which, in part, has to be related to post-chemotherapy consolidation treatment in patients with pelvis-confined disease (radiotherapy, 26%; total cystectomy, 11%). Fifteen patients died of chemotherapy-related complications and in 16% of the patients toxicity led to discontinuation of treatment. Modern cisplatin-based chemotherapy leads to long-term survival and cure of selected patients with advanced urothelial transitional cancer. In routine clinical practice, chemotherapy should be offered to good prognosis patients; those presenting with a good performance status and a non-metastasising T4b tumour or with metastases confined to lymph nodes. Post-chemotherapy consolidation treatment by surgery or radiotherapy should always be considered. Such chemotherapy requires oncological expertise in order to avoid unnecessary toxicity.
Piritrexim is a lipid-soluble drug which is as effective an inhibitor of dihydrofolate reductase as methotrexate. Phase I and II studies have indicated activity in some tumour types. Because of its lipophilicity we have conducted a phase II study in recurrent high-grade malignant glioma (grades III and IV). Twenty-seven patients were treated with 25 mg p.o. three times daily for five consecutive days, repeated weekly, with provision for dose escalation or reduction according to toxicity. Five patients received less than 4 weeks' treatment because of disease progression or death. Twenty-two patients were evaluable for response. One complete and one partial response was seen (duration 262+ and 241+ weeks) and 13 patients had static disease for a median duration of 13 weeks (range 7-35). The major toxicity was myelosuppression. This response rate of 9% of evaluable patients is much lower than that seen for some conventionally used drugs and we conclude that piritrexim is unlikely to be of value in the management of high-grade gliomas.
Piritrexim is a lipid-soluble inhibitor of dihydrofolate reductase (DHFR) that enters tumour cells rapidly by passive diffusion, cannot be polyglutamated, and is as effective as methotrexate in inhibiting DHFR. Bioavailability after oral dosing is approximately 75%. We performed a phase II study with oral piritrexim in non-chemotherapy pretreated patients with metastatic urothelial cancer. Thirty-three patients were treated with 25 mg three times daily for 5 consecutive days, repeated weekly, with provision for dose escalation or reduction according to the toxicity observed. Of 29 evaluable patients, one patient achieved a complete response of 19+ weeks duration, and ten patients achieved a partial response with a median duration of 22 weeks (range 16-48), for a total response rate of 38%. Piritrexim was generally well tolerated, with myelosuppression as the major toxicity, that frequently required dose modification. We conclude that piritrexim appears to be an active agent in patients with metastatic urothelial cancer when administered as a 5-day, low-dose oral schedule. It would be attractive to investigate the combination of piritrexim and cisplatin.
L-Glutamine is a requirement for many cells in tissue culture, an intermediate in many metabolic pathways, and an alternative substrate to glucose for energy metabolism. These properties suggest that glutamine concentration might be a determinant of cell viability in tumours, especially in regions that are deficient in other metabolites. We have therefore studied the effects of glutamine depletion on single cells in culture, on spheroids and on experimental tumours. Absence of glutamine suppressed the growth rate of two cell lines, but cells cultured for up to 6 h in the absence of glutamine had no decrease in plating efficiency. There was little effect on growth of MGH-U1 (human bladder cancer) spheroids of varying the glutamine concentration in the range of 0.1 to 2 mM and spheroids exposed to these concentrations did not develop central necrosis. Lower concentration of glutamine suppressed the rate of spheroid growth, and spheroids did not grow in the absence of glutamine. Pseudomonas 7A glutaminase reduced the survival of cells in glutamine-free culture and prevented growth of spheroids. Glutaminase was injected into mice bearing experimental tumours to reduce blood levels of glutamine; some animals also received 15 Gy radiation to their tumours to assess the effects of glutamine levels on surviving nutrient-deprived (i.e. hypoxic) cells. Glutaminase had no effect on cell survival in the Lewis lung tumour or in MGH-U1 xenografts, with or without radiation; glutaminase caused dose-dependent growth delay of the KHT tumour, which was additive to that caused by radiation. The present results suggest that (i) short-term changes of glutamine concentration have small effects on cell viability; and (ii) depletion of glutamine levels in blood through the in vivo use of glutaminase is unlikely to produce major therapeutic effects against nutrient-deprived cells in solid tumours.
A series of xenografts of human breast carcinomas has been established and serially transplanted in immune-suppressed mice. Certain structural and functional features of the original human tumours, including carcinoembryonic antigen and epithelial membrane antigen, continue to be expressed by the resulting xenografts. Stromal responses such as elastosis and oestrogen-receptor activity were lost by the xenografts. No metastases were detected in tumour-bearing mice. This study suggests that xenografts may have some value in experimental pathology as one type of model of human breast carcinoma.
Fifty-six patients with disseminated malignant melanoma were randomly allocated to two treatment groups. The first group C received combination chemotherapy consisting of DTIC and ICRF 159. The second group (C+I) received the same chemotherapy but were also immunized with 2 X 10(7) irradiated allogeneic melanoma cells mixed with 50 mug of percutaneous BCG. The survival rates in both treatment groups C and (C+I) were not significantly different, and only minor enhancement of the chemotherapy was found in the (C+I) group. A similar pattern of tissue response was observed in both groups: lymph node, skin and, to some extent liver metastases, respond better than other sites.
The early changes induced by a carcinogenic nitrosoquinoline compound (NTDQ) have been studied in the subcutaneous tissues of 88 rats. An initial acute infiammatory response is quickly replaced by a distinctive granuloma which is established by 10 days and persists indefinitely—a sequence which takes place both in adult and in newborn animals. Its main components—histiocytes, multinucleate giant cells and granulation tissue—are described in detail and the formation of giant cells by fusion from adjacent histiocytes has been traced. Autoradiographic studies with tritiated thymidine show heavy nuclear labelling in the histiocytes and fibroblasts during the first 10 days; this later declines but raised levels of nuclear labelling persist up to the end of the experiment. No proliferative activity is seen in the giant cells and these cells show only feeble phagocytic activity, tested by their ability to take up carbon particles. The experiments in which colloidal carbon was injected locally also provided some information on the lymphatic vessels in the vicinity of NTDQ-induced granulomata. It appears that, initially, the lesions contain large dilated lymphatic vessels. Later, a dense connective tissue barrier develops and lymphatic connections with the surrounding dermis are progressively reduced.
The properties of granulomata induced by NTDQ are discussed and some possible relationships between the formation of granulomata and eventual tumour developed are considered. Particular emphasis is given to two related features: the sustained proliferative activity of the fibroblasts and the resulting semi-isolation of the injection site lesion by the formation of a dense connective tissue barrier.