RAC1 activity is critical for intestinal homeostasis, and is required for hyperproliferation driven by loss of the tumour suppressor gene Apc in the murine intestine. To avoid the impact of direct targeting upon homeostasis, we reasoned that indirect targeting of RAC1 via RAC-GEFs might be effective. Transcriptional profiling of Apc deficient intestinal tissue identified Vav3 and Tiam1 as key targets. Deletion of these indicated that while TIAM1 deficiency could suppress Apc- driven hyperproliferation, it had no impact upon tumourigenesis, while VAV3 deficiency had no effect. Intriguingly, deletion of either gene resulted in upregulation of Vav2 , with subsequent targeting of all three ( Vav2 −/− Vav3 −/− Tiam1 −/− ), profoundly suppressing hyperproliferation, tumourigenesis and RAC1 activity, without impacting normal homeostasis. Critically, the observed RAC-GEF dependency was negated by oncogenic KRAS mutation. Together, these data demonstrate that while targeting RAC-GEF molecules may have therapeutic impact at early stages, this benefit may be lost in late stage disease.

KRAS is the most frequently mutated driver oncogene in human adenocarcinoma of the lung. There are presently no clinically proven strategies for treatment of KRAS-driven lung cancer. Activating mutations in KRAS are thought to confer independence from upstream signaling; however, recent data suggest that this independence may not be absolute. We show that initiation and progression of KRAS-driven lung tumors require input from ERBB family receptor tyrosine kinases (RTKs): Multiple ERBB RTKs are expressed and active from the earliest stages of KRAS-driven lung tumor development, and treatment with a multi-ERBB inhibitor suppresses formation of KRASG12D-driven lung tumors. We present evidence that ERBB activity amplifies signaling through the core RAS pathway, supporting proliferation of KRAS-mutant tumor cells in culture and progression to invasive disease in vivo. Brief pharmacological inhibition of the ERBB network enhances the therapeutic benefit of MEK (mitogen-activated protein kinase kinase) inhibition in an autochthonous tumor setting. Our data suggest that lung cancer patients with KRAS-driven disease may benefit from inclusion of multi-ERBB inhibitors in rationally designed treatment strategies.

NUAK1 is a member of the AMPK-related family of kinases. Recent evidence suggests that NUAK1 is an important regulator of cell adhesion and migration, cellular and organismal metabolism, and regulation of TAU stability. As such, NUAK1 may play key roles in multiple diseases ranging from neurodegeneration to diabetes and metastatic cancer. Previous work revealed a crucial role for NUAK1 in supporting viability of tumour cells specifically when MYC is overexpressed. This role is surprising, given that NUAK1 is activated by the tumour suppressor LKB1. Here we show that, in tumour cells lacking LKB1, NUAK1 activity is maintained by an alternative pathway involving calcium-dependent activation of PKCα. Calcium/PKCα-dependent activation of NUAK1 supports engagement of the AMPK-TORC1 metabolic checkpoint, thereby protecting tumour cells from MYC-driven cell death, and indeed, MYC selects for this pathway in part via transcriptional regulation of PKCα and ITPR. Our data point to a novel role for calcium in supporting tumour cell viability and clarify the synthetic lethal interaction between NUAK1 and MYC.

Introduction: The inevitability of tumour recurrence in glioblastoma (GBM) patients despite multi-modality treatment consisting of surgery, radiotherapy and chemotherapy, is reflected by a median survival of only 14 months. Tumour recurrence is thought to be driven by a small population of glioblastoma stem-like cells (GSCs) that are resistant to conventional therapies. DNA damage response (DDR) pathways have been shown to be up-regulated in GSCs and implicated in radioresistance and treatment failure. However the precise cause of enhanced DDR signalling in GSCs and the extent to which these signalling networks contribute to therapy resistance remains elusive. The objectives of this study were to investigate the underlying cause of DDR upregulation and treatment resistance in GSCs with a view to identifying novel and promising therapeutic targets. Materials and Methods: A panel of primary patient derived GBM cell lines cultured under conditions to enrich for or deplete the tumour stem cell population (GSC vs bulk respectively) were utilised in order to investigate enhanced GSC DDR under basal conditions and in response to ionising radiation. Confirmatory studies were also performed in cells sorted for the putative GSC marker CD133. The effects of a panel of small molecule DDR inhibitor agents on cell survival in GSC and bulk cells were quantified. Results: GSCs exhibited higher levels of total and activated DDR targets ATR, CHK1, ATM and PARP1 under basal conditions and were radioresistant compared to paired bulk populations. This was not due to increased levels of reactive oxygen species (ROS). Instead, we show that RPA is significantly higher in replicating GSCs and confirm by DNA fibre assays that GSCs and CD133+ cells have increased numbers of stalled replication forks, fewer new origins and slower DNA replication compared to bulk or CD133- populations, demonstrating for the first time that replication stress (RS) is a hallmark of GSCs. We identify increased expression of long neural genes as a likely mechanism for RS and DNA double strand breaks (DSBs) in GSCs and show that their radioresistance is reversed by dual inhibition of key RS and DDR proteins ATR and PARP. Conclusions: This study demonstrates the novel finding that replication stress is a hallmark of GSCs and resonates with recently published studies in neural progenitor cells showing that RS preferentially induces DNA DSB in long neural genes. Taken together, we implicate RS as a driver of enhanced DDR in GSCs and identify novel therapeutics with potential to improve clinical outcomes by overcoming the radioresistance of GBM

G12 mutant KRAS requires tonic ERBB network activity for initiation and maintenance of lung cancer.

Ketorolac tromethamine administered intramuscularly (i.m.) 10 mg 6-hourly was compared with morphine 10 mg i.m. 6-hourly in a randomised, double-blind, cross-over trial for its analgesic efficacy and safety in 51 patients with moderate to severe cancer pain. There was no overall significant difference between the analgesic effect of the two treatments. 57% of ketorolac- and 74% of morphine-treated patients changed their analgesic. Among these, significantly more patients stopped ketorolac than morphine due to pain (p=0.007) whilst more patients discontinued morphine than ketorolac because of adverse effects (p=0.001), predominantly emesis. Only one patient (2%) stopped ketorolac because of intolerance. Ketorolac shows promise as an effective and well tolerated analgesic for cancer pain and merits further study.

We report the use of the steroidal aromatase inhibitor, 4-hydroxyandrostenedione (4-OHA, CGP 32349), in the management of patients with advanced, hormone resistant, prostatic cancer. Eighteen of 25 patients (72%) showed a subjective response, mainly in the form of pain relief and increased performance. There were no objective improvements. A tumour flare occurred in 17/25 (68%). Detailed endocrine studies were performed during treatment. These showed that suppression of serum oestradiol levels occurred in 19/25 (76%) of patients during treatment with 4-OHA. Serum levels of androstenedione increased in 9/14 patients (64%). Concentration of serum testosterone and 5 alpha-dihydrotestosterone were elevated in 3/14 (21%) and 2/11 (18%) patients respectively. There appeared to be no correlation between response or tumour flare and changes in steroid levels during treatment with 4-OHA. The mechanism of action of 4-OHA in palliating patients with advanced prostatic cancer remains obscure. 4-OHA or its metabolites may be acting on metastatic bone metabolism via effects on oestrogen related osteoclastic and osteoblastic activity. Further investigation of the effects of aromatase inhibitors on prostatic biology, and bone metabolism in patients with metastatic prostate cancer, would appear worthwhile.

A double-blind placebo-controlled trial was undertaken to assess the value of topical metronidazole gel preparation in the palliation of the offensive odour of fungating tumours. This 5 day trial was followed by a 6 day period during which all patients received the active gel. Subjective odour assessments were performed by both patients and medical staff. 11 patients were entered and the randomisation was then halted because of an obvious overall benefit over the whole 11 days. There was a non-significant trend in favour of the active treatment during the initial double-blind placebo-controlled phase of the trial, and no side-effects were observed.

19 patients with locally advanced or cutaneous metastatic breast cancer were treated with the topical vitamin D analogue calcipotriol 100 μg daily. 14 patients completed 6 weeks' treatment; 3 showed a 50% reduction in the bidimensional diameter of treated lesions and 1 other patient showed a minimal response. 2 patients became hypercalcaemic during treatment. In all patients who responded the tumours contained receptors for 1,25-dihydroxyvitamin D3, shown by immunocytochemistry.

Thirty-one postmenopausal women with advanced breast cancer have been treated with the nonsteroidal competitive aromatase inhibitor CGS 16949A at p.o. doses of 0.3, 1, and 2 mg twice a day. All patients were assessed for response. Five patients, all treated with 1 mg twice daily, had objective evidence of response (two complete responses and three partial responses); disease stabilized in 17 patients. Minor side effects were reported by ten patients. Two further patients treated with 2 mg twice a day experienced persistent nausea which improved after dose reduction, and one patient, treated with 0.3 mg twice daily, developed a vasculitic rash requiring discontinuation of CGS 16949A. Estradiol levels measured in 24 patients were significantly suppressed 2 wk after starting CGS 16949A treatment at all doses used. Treatment with 2 mg twice a day lowered estradiol levels to a mean of 29% of pretreatment values which was significantly lower than the corresponding figure of 57% for patients treated with 0.3 mg twice daily. Aldosterone levels were significantly lowered below pretreatment values by the 1- and 2-mg twice daily doses. No clinically apparent cases of adrenocortical insufficiency occurred, although small changes in serum electrolyte levels were noted. The results indicate that CGS 16949A is an effective aromatase inhibitor, requiring further evaluation in the treatment of advanced breast cancer. The optimal dose is likely to be 1 mg twice a day.

The aromatase inhibitor, 4-hydroxyandrostenedione (4OHA) is an effective treatment for advanced post-menopausal breast cancer. The clinical and endocrine effects of 4OHA treatment were studied in five pre- and perimenopausal women with metastatic breast cancer. Serum oestradiol levels were not significantly reduced as a result of treatment with 500 mg of 4OHA by weekly i.m. injections and no patient had a tumour response. Four patients were subsequently treated with the luteinising hormone releasing hormone (LHRH) analogue, gosereline, and three had objective responses. The endocrine effects of combined treatment with goserelin (Zoladex), and 4OHA were studied in a further five premenopausal women. Serum oestradiol levels after treatment with goserelin alone were typical of post-menopausal women. Addition of 4OHA led to a further suppression of oestradiol to within the range observed in post-menopausal patients treated with further suppression of oestradiol to within the range observed in post-menopausal patients treated with 4OHA. Six patients whose tumours had regressed as a result of goserelin treatment and who subsequently relapsed were then given combined treatment. Four of the six experienced a second remission. We conclude that while 4OHA alone is unlikely to be a satisfactory treatment for premenopausal patients with advanced breast cancer, 4OHA in conjunction with goserelin leads to profound suppression of oestradiol. The combination of LHRH analogue and aromatase inhibitor may prove to be a superior treatment to LHRH analogue alone in these patients.

We report the results of the first use of a steroidal aromatase inhibitor, 4-hydroxyandrostenedione (4-OHA, CGP 32349), in the palliation of patients with advanced, hormone resistant, prostatic cancer. Twelve of 19 patients (63%), who had relapsed following castration and other therapies, gained significant pain relief following weekly intramuscular injections of 4-OHA. Five patients (31%) experienced a transient 'tumour flare', represented by an increase in bone pain soon after commencing treatment. The mechanism of action of 4-OHA in palliating patients with advanced prostatic cancer is obscure at present, but may represent an important new treatment modality which may lead to greater insight into prostatic biology.

4-Hydroxyandrostenedione (CGP32349; 4-OHA) is a clinically effective treatment for advanced postmenopausal breast cancer by both the parenteral and p.o. routes, as a result of its inhibition of aromatase and consequent suppression of plasma estrogen levels. Thirty patients were randomized to treatment with 250 mg 4-OHA orally once, twice, and 4 times daily for 2 weeks and 29 of these plus a further 11 patients were then randomized to treatment with 250 or 500 mg i.m. every 2 weeks to determine the optimal dose for each route according to the suppression of serum estradiol levels. There was no significant difference between the 3 oral doses in their suppression of estradiol levels indicating that the maximum required p.o. dose of 4-OHA is probably 250 mg daily. Suppression by the parenteral dose of 250 mg every 2 weeks was marginally suboptimal but clinical considerations of response and tolerability indicate this as the optimal dose for i.m. injection. 4-OHA had no effect on serum levels of androstenedione, testosterone, or 5 alpha-dihydrotestosterone when given by either route but p.o. treatment with 4 doses of 250 mg daily reduced sex hormone-binding globulin levels by a mean of 34%. Serum levels of estrone as measured by gas chromatography-mass spectrometry were suppressed to approximately 40% of baseline by parenteral treatment. The half-life of 4-OHA p.o. was approximately 3 h, whereas the apparent half-life of injected drug was between 5 and 10 days after a more rapid clearance during the first 4 days after injection.