Acquired resistance to temsirolimus in human renal cell carcinoma cells is mediated by the constitutive activation of signal transduction pathways through mTORC2

Background:

The objective of this study was to characterise the mechanism underlying acquired resistance to temsirolimus, an inhibitor of mammalian target of rapamycin (mTOR), in renal cell carcinoma (RCC).

Methods:

A parental human RCC cell line, ACHN (ACHN/P), was continuously exposed to increasing doses of up to 20 μM of temsirolimus, and a cell line resistant to temsirolimus (ACHN/R), showing a sixfold higher IC₅₀ than that of ACHN/P, was developed.

Results:

Following treatment with temsirolimus, phosphorylation of S6 kinase in ACHN/P was markedly inhibited, whereas there was no detectable expression of phosphorylated S6 in ACHN/R before and after temsirolimus treatment. However, AKT and p44/42 mitogen-activated protein kinase (MAPK) were constitutively phosphorylated even after temsirolimus treatment in ACHN/R, but not in ACHN/P. There was no significant difference between the sensitivities of ACHN/P and ACHN/R to KU0063794, a dual inhibitor of mTOR complex 1 (mTORC1) and mTORC2. Similar sensitivities to temsirolimus in ACHN/P and ACHN/R could be achieved by additional treatment with specific inhibitors of AKT- and MAPK-signaling pathways.

Conclusion:

The activation of signal transduction pathways via mTORC2, but not via mTORC1, may have an important role in the acquisition of a resistant phenotype to temsirolimus in RCC.     

Tumour hypoxia determines the potential of combining mTOR and autophagy inhibitors to treat mammary tumours

Background:

Hypoxia can activate autophagy, a self-digest adaptive process that maintains cell turnover. Mammalian target of rapamycin (mTOR) inhibitors are used to treat cancer but also stimulate autophagy.

Methods:

Human mammary cancer cells and derived xenografts were used to examine whether hypoxia could exacerbate autophagy-mediated resistance to the mTOR inhibitor rapamycin.

Results:

Rapamycin exerted potent antitumour effects in MCF-7 and MDA-MB-231 mammary tumours through a marked inhibition of angiogenesis, but the autophagy inhibitor chloroquine (CQ) failed to further sensitise tumours to mTOR inhibition. Rapamycin treatment actually led to tumour reoxygenation, thereby preventing the development of autophagy. Chloroquine alone, however, blocked the growth of MCF-7 tumours and in vitro blunted the hypoxia-induced component of autophagy in these cells. Finally, when initiating CQ treatment in large, hypoxic tumours, a robust antitumour effect could be observed, which also further increased the antiproliferative effects of rapamycin.

Conclusion:

The mTOR inhibitor rapamycin significantly contributes to tumour growth inhibition and normalisation of the tumour vasculature through potent antiangiogenic effects. The resulting reduction in hypoxia accounts for a lack of sensitisation by the autophagy inhibitor CQ, except if the tumours are already at an advanced stage, and thus largely hypoxic at the initiation of the combination of rapamycin and CQ treatment.     

Integrated preclinical and clinical development of mTOR inhibitors in pancreatic cancer

Background:

The purpose of this work was to determine the efficacy of inhibiting mammalian target of rapamycin (mTOR) in pancreatic cancer preclinical models and translate preclinical observations to the clinic.

Methods:

Temsirolimus (20 mg Kg⁻¹ daily) was administered to freshly generated pancreatic cancer xenografts. Tumour growth inhibition was determined after 28 days. Xenografts were characterised at baseline by gene expression and comparative genomic hybridisation. Patients with advanced, gemcitabine-resistant pancreatic cancer were treated with sirolimus (5 mg daily). The primary end point was 6-month survival rate (6mSR). Correlative studies included immunohistochemistry assessment of pathway expression in baseline tumours, drug pharmacokinetics (PKs), response assessment by FDG-PET and pharmacodynamic effects in peripheral-blood mononuclear cells (PBMCs).

Results:

In all, 4 of 17 xenografts (23%) responded to treatment. Sensitive tumours were characterised by gene copy number variations and overexpression of genes leading to activation of the PI3K/Akt/mTOR pathway. Activation of p70S6K correlated with drug activity in the preclinical studies. Sirolimus was well tolerated in the clinic, showed predictable PKs, exerted pathway inhibition in post-treatment PBMCs and resulted in a 6mSR of 26%. No correlation, however, was found between activated p70S6K in tumour tissues and anti-tumour effects.

Conclusion:

Sirolimus activity in pancreatic cancer was marginal and not predicted by the selected biomarker.     

A Phase I Study of Temsirolimus and Bryostatin‐1 in Patients With Metastatic Renal Cell Carcinoma and Soft Tissue Sarcoma

Background.

Temsirolimus, an inhibitor of mammalian target of rapamycin (mTOR) complex 1, is approved for the treatment of metastatic renal cell carcinoma (RCC). Bryostatin-1 inhibits protein kinase C, a downstream effector of mTOR complex 2. We observed antitumor effects with the combination of temsirolimus and bryostatin-1 in RCC cell lines.

Methods.

Four cohorts of patients received weekly bryostatin-1 (20 μg/m²) with temsirolimus (10, 15, 25, or 37.5 mg) in 28-day cycles.

Results.

Thirty patients received a total of 138 cycles across four dose levels. Twenty-five patients had RCC (17 clear cell, 7 papillary, and 1 unclassified). Two sarcoma patients with prior cytotoxic therapy experienced dose-limiting toxicity at 15 mg of temsirolimus (grade 3 neutropenia and grade 3 hypophosphatemia). Subsequently, patients with prior cytotoxic therapy were excluded. Two additional dose-limiting toxicities were noted with 37.5 mg of temsirolimus (grade 3 neutropenia and grade 3 creatinine elevation). Consequently, the maximum tolerated dose was defined as temsirolimus at 25 mg and bryostatin-1 at 20 μg/m² every 28 days. Of the 25 RCC patients, 3 patients had partial responses that lasted for 14 months, 28 months, and ≥80 months, respectively. Partial responses were seen in both clear cell and papillary histology.

Conclusion.

This combination of 37.5 mg of temsirolimus with 20 μg/m² of bryostatin-1 was reasonably safe and well tolerated. Durable responses were observed in 3 of 25 patients with RCC.     

Aberrant activation of the mTOR pathway and anti-tumour effect of everolimus on oesophageal squamous cell carcinoma

Background:

The mammalian target of rapamycin (mTOR) protein is important for cellular growth and homeostasis. The presence and prognostic significance of inappropriate mTOR activation have been reported for several cancers. Mammalian target of rapamycin inhibitors, such as everolimus (RAD001), are in development and show promise as anti-cancer drugs; however, the therapeutic effect of everolimus on oesophageal squamous cell carcinoma (OSCC) remains unknown.

Methods:

Phosphorylation of mTOR (p-mTOR) was evaluated in 167 resected OSCC tumours and 5 OSCC cell lines. The effects of everolimus on the OSCC cell lines TE4 and TE11 in vitro and alone or in combination with cisplatin on tumour growth in vivo were evaluated.

Results:

Mammalian target of rapamycin phosphorylation was detected in 116 tumours (69.5%) and all the 5 OSCC cell lines. Everolimus suppressed p-mTOR downstream pathways, inhibited proliferation and invasion, and induced apoptosis in both TE4 and TE11 cells. In a mouse xenograft model established with TE4 and TE11 cells, everolimus alone or in combination with cisplatin inhibited tumour growth.

Conclusion:

The mTOR pathway was aberrantly activated in most OSCC tumours. Everolimus had a therapeutic effect both as a single agent and in combination with cisplatin. Everolimus could be a useful anti-cancer drug for patients with OSCC.     

A serum metabolomic fingerprint of bevacizumab and temsirolimus combination as first-line treatment of metastatic renal cell carcinoma

Background:

Renal cell carcinoma is one of the most chemoresistant cancers, and its metastatic form requires administration of targeted therapies based on angiogenesis or mTOR inhibitors. Understanding how these treatments impact the human metabolism is essential to predict the host response and adjust personalised therapies. We present a metabolomic investigation of serum samples from patients with metastatic RCC (mRCC) to identify metabolic signatures associated with targeted therapies.

Methods:

Pre-treatment and serial on-treatment sera were available for 121 patients participating in the French clinical trial TORAVA, in which 171 randomised patients with mRCC received a bevacizumab and temsirolimus combination (experimental arm A) or a standard treatment: either sunitinib (B) or interferon-α+bevacizumab (C). Metabolic profiles were obtained using nuclear magnetic resonance spectroscopy and compared on-treatment or between treatments.

Results:

Multivariate statistical modelling discriminates serum profiles before and after several weeks of treatment for arms A and C. The combination A causes faster changes in patient metabolism than treatment C, detectable after only 2 weeks of treatment. Metabolites related to the discrimination include lipids and carbohydrates, consistently with the known RCC metabolism and side effects of the drugs involved. Comparison of the metabolic profiles for the three arms shows that temsirolimus, an mTOR inhibitor, is responsible for the faster host metabolism modification observed in the experimental arm.

Conclusions:

In mRCC, metabolomics shows a faster host metabolism modification induced by a mTOR inhibitor as compared with standard treatments. These results should be confirmed in larger cohorts and other cancer types.     

Risk of infections in renal cell carcinoma (RCC) and non-RCC patients treated with mammalian target of rapamycin inhibitors

Background:

Mammalian target of rapamycin (mTOR) inhibitors are used in a variety of malignancies. Infections have been reported with these drugs. We performed an up-to-date meta-analysis to further characterise the risk of infections in cancer patients treated with these agents.

Methods:

Pubmed and oncology conferences'' proceedings were searched for studies from January 1966 to June 2012. Studies were limited to phase II and III randomised controlled trials (RCTs) of everolimus or temsirolimus reporting on cancer patients with adequate safety profiles. Summary incidences, relative risks (RRs), and 95% confidence intervals (CIs) were calculated.

Results:

A total of 3180 patients were included. The incidence of all-grade and high-grade infections due to mTOR inhibitors was 33.1% (95% CI, 24.5–43.0%) and 5.6% (95% CI, 3.8–8.3%), respectively. Compared with controls, the RR of all-grade and high-grade infections due to mTOR inhibitors was 2.00 (95% CI, 1.76–2.28, P<0.001) and 2.60 (95% CI, 1.54–4.41, P<0.001), respectively. Subgroup analysis found no difference in incidences or risks between everolimus and temsirolimus or between different tumour types (renal cell carcinoma (RCC) vs non-RCC). Infections included respiratory tract (61.7%), genitourinary (29.4%), skin/soft tissue (4.2%), and others (4.9%).

Conclusion:

Treatment with mTOR inhibitors is associated with a significant increase in risk of infections. Close monitoring for any signs of infections is warranted.     

PG545, a dual heparanase and angiogenesis inhibitor, induces potent anti-tumour and anti-metastatic efficacy in preclinical models

Background:

PG545 is a heparan sulfate (HS) mimetic that inhibits tumour angiogenesis by sequestering angiogenic growth factors in the extracellular matrix (ECM), thus limiting subsequent binding to receptors. Importantly, PG545 also inhibits heparanase, the only endoglycosidase which cleaves HS chains in the ECM. The aim of the study was to assess PG545 in various solid tumour and metastasis models.

Methods:

The anti-angiogenic, anti-tumour and anti-metastatic properties of PG545 were assessed using in vivo angiogenesis, solid tumour and metastasis models. Pharmacokinetic (PK) data were also generated in tumour-bearing mice to gain an understanding of optimal dosing schedules and regimens.

Results:

PG545 was shown to inhibit angiogenesis in vivo and induce anti-tumour or anti-metastatic effects in murine models of breast, prostate, liver, lung, colon, head and neck cancers and melanoma. Enhanced anti-tumour activity was also noted when used in combination with sorafenib in a liver cancer model. PK data revealed that the half-life of PG545 was relatively long, with pharmacologically relevant concentrations of radiolabeled PG545 observed in liver tumours.

Conclusion:

PG545 is a new anti-angiogenic clinical candidate for cancer therapy. The anti-metastatic property of PG545, likely due to the inhibition of heparanase, may prove to be a critical attribute as the compound enters phase I clinical trials.     

Insulin growth factor receptor (IGF-1R) antibody cixutumumab combined with the mTOR inhibitor temsirolimus in patients with metastatic adrenocortical carcinoma

Background:

Adrenocortical carcinoma (ACC) is a rare and aggressive endocrine malignancy without an available effective systemic chemotherapy. Insulin growth factor 2 (IGF-2) overexpression leading to the activation of the IGF-1 receptor (IGF-1R)/mammalian target of rapamycin (mTOR) pathway is well described in ACC. Cixutumumab, a fully human IgG1 monoclonal antibody directed at IGF-1R was combined with temsirolimus on the basis of preclinical data.

Methods:

Patients received cixutumumab, 3–6 mg kg⁻¹ intravenously (IV) weekly, and temsirolimus, 25–37.5 mg IV weekly (4-week cycles), with restaging after 8 weeks.

Results:

Twenty-six patients were enrolled (13 (50%) men); median age, 47 years; median number of prior therapies, 4. Five patients previously received an IGF-1R inhibitor and one, temsirolimus. The most frequent toxicities, at least possibly drug related, were grade 1–2 thrombocytopenia (38%), mucositis (58%), hypercholesterolaemia (31%), hypertriglyceridemia (35%), and hyperglycaemia (31%). In all, 11 of 26 patients (42%) achieved stable disease (SD) >6 months (duration range=6–21 months) with 3 of the 11 having received a prior IGF-1R inhibitor.

Conclusion:

Cixutumumab combined with temsirolimus was well tolerated and >40% of patients achieved prolonged SD.     

Effects of preset sequential administrations of sunitinib and everolimus on tumour differentiation in Caki-1 renal cell carcinoma

Background:

Sunitinib (VEGFR/PDGFR inhibitor) and everolimus (mTOR inhibitor) are both approved for advanced renal cell carcinoma (RCC) as first-line and second-line therapy, respectively. In the clinics, sunitinib treatment is limited by the emergence of acquired resistance, leading to a switch to second-line treatment at progression, often based on everolimus. No data have been yet generated on programmed alternating sequential strategies combining alternative use of sunitinib and everolimus before progression. Such strategy is expected to delay the emergence of acquired resistance and improve tumour control. The aim of our study was to assess the changes in tumours induced by three different sequences administration of sunitinib and everolimus.

Methods:

In human Caki-1 RCC xenograft model, sunitinib was alternated with everolimus every week, every 2 weeks, or every 3 weeks. Effects on necrosis, hypoxia, angiogenesis, and EMT status were assessed by immunohisochemistry and immunofluorescence.

Results:

Sunitinib and everolimus programmed sequential regimens before progression yielded longer median time to tumour progression than sunitinib and everolimus monotherapies. In each group of treatment, tumour growth control was associated with inhibition of mTOR pathway and changes from a mesenchymal towards an epithelial phenotype, with a decrease in vimentin and an increase in E-cadherin expression. The sequential combinations of these two agents in a RCC mouse clinical trial induced antiangiogenic effects, leading to tumour necrosis.

Conclusions:

In summary, our study showed that alternate sequence of sunitinib and everolimus mitigated the development of mesenchymal phenotype compared with sunitinib as single agent.     

Safety and efficacy of the combination of erlotinib and sirolimus for the treatment of metastatic renal cell carcinoma after failure of sunitinib or sorafenib

Background:

The mammalian target of rapamycin (mTOR) is an important therapeutic target in the treatment of renal cell carcinoma (RCC). Pre-clinical data indicate that the combined inhibition of both the epidermal growth factor receptor and mTOR results in enhanced anticancer activity.

Methods:

All patients had metastatic RCC with progression after treatment with sunitinib and/or sorafenib. Treatment consisted of erlotinib 150 mg orally once a day starting on day 1 and sirolimus 6 mg orally on day 8 followed by 2 mg daily, adjusted according to blood levels.

Results:

A total of 25 patients were enrolled between July 2006 and March 2008. The median progression-free survival (PFS) was 12 weeks (95% CI 5.9–18.1) and median overall survival (OS) 40 weeks (95% CI 0–85.7). No confirmed complete or partial responses were observed, but stable disease >6 months was noted in 21.8% (95% CI 4.9–38.6) of patients. The most common adverse events were rash and diarrhoea. There was no correlation between erlotinib, OSI-420 (days 8 and 15) or sirolimus (days 15 and 29) blood levels and PFS or OS.

Conclusions:

The combination of sirolimus and erlotinib for RCC failed to demonstrate an advantage over available single-agent therapy in the second-line setting.     

A phase I study of E7080, a multitargeted tyrosine kinase inhibitor, in patients with advanced solid tumours

Background:

The objectives of this phase I study were to assess the safety and tolerability of E7080 in patients with advanced, refractory solid tumours; to determine the maximum tolerated dose (MTD) and pharmacokinetics profile of E7080; and to explore preliminary evidence of its anti-tumour efficacy.

Methods:

E7080 was administered orally in escalating doses on a once-daily continuous schedule in 28-day cycles to eligible patients. Samples for pharmacokinetic analyses were collected on days 1, 8, 15 and 22 of cycle 1 and day 1 of cycle 2. Anti-tumour efficacy was assessed every two cycles.

Results:

Eighty-two patients received E7080 in dose cohorts from 0.2 to 32 mg. Dose-limiting toxicities were grade 3 proteinuria (two patients) at 32 mg, and the MTD was defined as 25 mg. The most frequently observed cumulative toxicities (all grades) were hypertension (40% of patients), diarrhoea (45%), nausea (37%), stomatitis (32%) and vomiting (23%). Seven patients (9%) had a partial response and 38 patients (46%) had stable disease as best response. E7080 has dose-linear kinetics with no drug accumulation after 4 weeks'' administration.

Conclusion:

E7080 is well tolerated at doses up to 25 mg per day. Encouraging anti-tumour efficacy was observed in patients with melanoma and renal cell carcinoma.     

PTEN deficiency is associated with reduced sensitivity to mTOR inhibitor in human bladder cancer through the unhampered feedback loop driving PI3K/Akt activation

Background:

Preclinical studies have shown that PTEN loss enhances sensitivity to mammalian target of Rapamycin (mTOR) inhibitors because of facilitated PI3K (phosphatidylinositol-3 kinase)/Akt activation and consecutive stimulation of the mTOR pathway. In patients with advanced transitional cell carcinoma (TCC) treated with the mTOR inhibitor everolimus, PTEN loss was, however, associated with resistance to treatment.

Methods:

Transitional cell carcinoma specimens, human bladder cancer cells and derived mouse xenografts were used to evaluate how the PTEN status influences the activity of mTOR inhibitors.

Results:

Transitional cell carcinoma patients with a shorter progression-free survival under everolimus exhibited PTEN deficiency and increased Akt activation. Moreover, PTEN-deficient bladder cancer cells were less sensitive to rapamycin than cells expressing wild-type PTEN, and rapamycin strikingly induced Akt activation in the absence of functional PTEN. Inhibition of Akt activation by the PI3K inhibitor wortmannin interrupted this rapamycin-induced feedback loop, thereby enhancing the antiproliferative effects of the mTOR inhibitor both in vitro and in vivo.

Conclusion:

Facilitation of Akt activation upon PTEN loss can have a more prominent role in driving the feedback loop in response to mTOR inhibition than in promoting the mTOR pathway. These data support the use of both PI3K and mTOR inhibitors to treat urothelial carcinoma, in particular in the absence of functional PTEN.     

Phase I clinical trial of human vascular endothelial growth factor receptor 1 peptide vaccines for patients with metastatic renal cell carcinoma

Background:

It is well known that renal cell carcinoma (RCC) represents one of the most immune-responsive cancers. Although the lack of defined antigens in RCC has hindered more specific vaccine development, research regarding vaccination therapy has been of special interest for the treatment of RCC for more than 30 years.

Methods:

To evaluate the safety of the vascular endothelial growth factor receptor 1 (VEGFR1) peptide vaccination and its clinical outcomes, data from 18 metastatic RCC (mRCC) patients treated with VEGFR1 vaccine were collected. Toxicity assessments were performed. Clinical outcomes included assessment using CT scanning, magnetic resonance imaging or X-ray examination in accordance with the WHO Response Evaluation Criteria in Solid Tumors.

Results:

No patient showed any toxicities of grade 3 or greater. Of the 18 patients, 2 patients showed a partial response during treatment. Stable disease for more than 5 months was observed in eight patients with a median duration of 16.5 months (4–32 months). At the time of the analysis in this study, six patients were alive with a median follow-up of 30 months (26–36 months).

Conclusion:

These results suggest that VEGFR1 peptide vaccine is safe and is recommended for further trials for patients with mRCC.     

Inhibition of angiogenic and non-angiogenic targets by sorafenib in renal cell carcinoma (RCC) in a RCC xenograft model

Background:

It is widely recognised that sorafenib inhibits a range of molecular targets in renal cell carcinoma (RCC). In this study, we aim to use patient-derived RCC xenografts to delineate the angiogenic and non-angiogenic molecular targets of sorafenib therapy for advanced RCC (aRCC).

Methods:

We successfully generated three patient RCC-derived xenografts in severe combined immunodeficient mice, consisting of three different RCC histological subtypes: conventional clear cell, poorly differentiated clear cell RCC with sarcomatoid changes, and papillary RCC. This study also used clear cell RCC cells (786-0/EV) harbouring mutant VHL to investigate the clonogenic survival of cells transfected with survivin sense and antisense oligonucleotides.

Results:

All three xenografts retain their original histological characteristics. We reported that sorafenib inhibited all three RCC xenograft lines regardless of histological subtypes in a dose-dependant manner. Sorafenib-induced growth suppression was associated with not only inhibition of angiogenic targets p-PDGFR-β, p-VEGFR-2, and their downstream signalling pathways p-Akt and p-ERK, cell cycle, and anti-apoptotic proteins that include cyclin D1, cyclin B1, and survivin but also upregulation of proapoptotic Bim. Survivin knockdown by survivin-specific antisense-oligonucleotides inhibited colony formation and induced cell death in clear cell RCC cells.

Conclusion:

This study has shed light on the molecular mechanisms of sorafenib in RCC. Inhibition of non-angiogenic molecules by sorafenib could contribute in part to its anti-tumour activities observed in vivo, in addition to its anti-angiogenic effects.     

Immune activation by combination human lymphokine-activated killer and dendritic cell therapy

Background:

Optimal cellular immunotherapy for cancer should ideally harness both the innate and adaptive arms of the immune response. Lymphokine-activated killer cells (LAKs) can trigger early innate killing of tumour targets, whereas long-term adaptive-specific tumour control requires priming of CD8+ cytotoxic lymphocytes (CTLs) following acquisition of tumour-associated antigens (TAAs) by antigen-presenting cells such as dendritic cells (DCs). As DCs stimulate both innate and adaptive effectors, combination cell therapy using LAKs and DCs has the potential to maximise anti-tumour immune priming.

Methods:

Reciprocal activation between human clinical grade LAKs and DCs on co-culture, and its immune consequences, was monitored by cell phenotype, cytokine release and priming of both innate and adaptive cytotoxicity against melanoma targets.

Results:

Co-culture of DCs and LAKs led to phenotypic activation of natural killer (NK) cells within the LAK population, which was associated with increased production of inflammatory cytokines and enhanced innate cytotoxicity against tumour cell targets. The LAKs reciprocally matured DCs, and the combination of LAKs and DCs, on addition of melanoma cells, supported priming of specific anti-tumour CTLs better than DCs alone.

Conclusion:

Clinical-grade LAKs/DCs represents a practical, effective combination cell immunotherapy for stimulation of both innate and adaptive anti-tumour immunity in cancer patients.     

Long-term dietary sodium,potassium and fluid intake; exploring potential novel risk factors for renal cell cancer in the Netherlands Cohort Study on diet and cancer

Background:

As sodium, potassium and fluid intake are related to hypertension, an established risk factor for renal cell cancer (RCC), they may be independent risk factors for RCC.

Methods:

The Netherlands Cohort Study (NLCS) with case-cohort design included 120 852 participants aged 55–69 years. At baseline, diet and lifestyle were assessed with questionnaires. After 17.3 years of follow-up, 485 RCC cases and 4438 subcohort members were available for analyses.

Results:

Sodium intake increased RCC risk (P-trend=0.03), whereas fluid and potassium intake did not. For high sodium and low fluid intake, the RCC risk additionally increased (P-interaction=0.02).

Conclusion:

Sodium intake is a potential risk factor for RCC, particularly if fluid consumption is low.     

Axl receptor tyrosine kinase is a potential therapeutic target in renal cell carcinoma

Background:

Axl plays multiple roles in tumourigenesis in several cancers. Here we evaluated the expression and biological function of Axl in renal cell carcinoma (RCC).

Methods:

Axl expression was analysed in a tissue microarray of 174 RCC samples by immunostaining and a panel of 11 normal tumour pairs of human RCC tissues by western blot, as well as in RCC cell lines by both western blot and quantitative PCR. The effects of Axl knockdown in RCC cells on cell growth and signalling were investigated. The efficacy of a humanised Axl targeting monoclonal antibody hMAb173 was tested in histoculture and tumour xenograft.

Results:

We have determined by immunohistochemistry (IHC) that Axl is expressed in 59% of RCC array samples with moderate to high in 20% but not expressed in normal kidney tissue. Western blot analysis of 11 pairs of tumour and adjacent normal tissue show high Axl expression in 73% of the tumours but not normal tissue. Axl is also expressed in RCC cell lines in which Axl knockdown reduces cell viability and PI3K/Akt signalling. The Axl antibody hMAb173 significantly induced RCC cell apoptosis in histoculture and inhibited the growth of RCC tumour in vivo by 78%. The hMAb173-treated tumours also had significantly reduced Axl protein levels, inhibited PI3K signalling, decreased proliferation, and induced apoptosis.

Conclusions:

Axl is highly expressed in RCC and critical for RCC cell survival. Targeting Axl is a potential approach for RCC treatment.     

mTOR inhibition improves fibroblast growth factor receptor targeting in hepatocellular carcinoma

Background:

Systemic therapy has proven only marginal effects in hepatocellular carcinoma (HCC) so far. The aim of this study was to evaluate the effect of targeting fibroblast growth factor receptor (FGFR) on tumour and stromal cells in HCC models.

Methods:

Human and murine HCC cells, endothelial cells (ECs), vascular smooth muscle cells (VSMCs), hepatic stellate cells (HSCs), human HCC samples, FGFR inhibitor BGJ398 and mammalian target of rapamycin (mTOR) inhibitor rapamycin were used. Effects on growth, motility, signalling and angiogenic markers were determined. In vivo subcutaneous and syngeneic orthotopic tumour models were used.

Results:

In tumour cells and ECs, targeting FGFR showed significant inhibitory effects on signalling and motility. Minor effects of FGFR inhibition were observed on VSMCs and HSCs, which were significantly enhanced by combining FGFR and mTOR blockade. In vivo daily (5 mg kg⁻¹) treatment with BGJ398 led to a significant growth inhibition in subcutaneous tumour models, but only a combination of FGFR and mTOR blockade impaired tumour growth in the orthotopic model. This was paralleled by reduced tumour cell proliferation, vascularisation, pericytes and increased apoptosis.

Conclusions:

Targeting FGFR with BGJ398 affects tumour cells and ECs, whereas only a combination with mTOR inhibition impairs recruitment of VSMCs and HSCs. Therefore, this study provides evidence for combined FGFR/mTOR inhibition in HCC.     

Prolonging hormone sensitivity in prostate cancer xenografts through dual inhibition of AR and mTOR

Background:

To determine the mechanisms associated with loss of androgen dependency and disease progression in prostate cancer (PCa), we investigated the relationship between the androgen receptor (AR) and mTOR pathways and the impact of inhibiting both pathways in androgen-dependent and castration-resistant PCa models.

Experimental design:

Androgen-dependent (LNCaP) and castration-resistant PCa (HP-LNCaP) cells were grown as tumours in SCID mice. Once tumours reached 500 mm³, animals were grouped and injected subcutaneous with vehicle, our novel anti-androgen/androgen synthesis inhibitor, VN/124-1, bicalutamide, and everolimus. Tumour volumes were measured biweekly. The PSA and protein analyses were performed after completion of the treatment.

Results:

The addition of everolimus to bicalutamide treatment of resistant tumours significantly reduced tumour growth rates and tumour volumes. Anti-androgen treatment also increased protein expression of multiple signal transduction pathways earlier than vehicle-treated control xenografts. VN/124-1 plus everolimus acted in concert to reduce tumour growth rates in our castration-resistant xenograft model.

Conclusions:

This study suggests that dual inhibition of AR and mTOR in castration-resistant xenograft models can restore sensitivity of tumours to anti-androgen therapy. Furthermore, after bicalutamide failure, dual inhibition with VN/124-1 and everolimus was the most effective treatment.