iRhom2 regulates ERBB signalling to promote KRAS-driven tumour growth of lung cancer cells.

Dysregulation of the ERBB/EGFR signalling pathway causes multiple types of cancer. Accordingly, ADAM17, the primary shedding enzyme that releases and activates ERBB ligands, is tightly regulated. It has recently become clear that iRhom proteins, inactive members of the rhomboid-like superfamily, are regulatory cofactors for ADAM17. Here, we show that oncogenic KRAS mutants target the cytoplasmic domain of iRhom2 (also known as RHBDF2) to induce ADAM17-dependent shedding and the release of ERBB ligands. Activation of ERK1/2 by oncogenic KRAS induces the phosphorylation of iRhom2, recruitment of the phospho-binding 14-3-3 proteins, and consequent ADAM17-dependent shedding of ERBB ligands. In addition, cancer-associated mutations in iRhom2 act as sensitisers in this pathway by further increasing KRAS-induced shedding of ERBB ligands. This mechanism is conserved in lung cancer cells, where iRhom activity is required for tumour xenograft growth. In this context, the activity of oncogenic KRAS is modulated by the iRhom2-dependent release of ERBB ligands, thus placing the cytoplasmic domain of iRhom2 as a central component of a positive feedback loop in lung cancer cells. This article has an associated First Person interview with the first authors of the paper.

Beyond cancer cells: Targeting the tumor microenvironment with gene therapy and armed oncolytic virus.

Cancer gene therapies are usually designed either to express wild-type copies of tumor suppressor genes or to exploit tumor-associated phenotypic changes to endow selective cytotoxicity. However, these approaches become less relevant to cancers that contain many independent mutations, and the situation is made more complex by our increased understanding of clonal evolution of tumors, meaning that different metastases and even regions of the same tumor mass have distinct mutational and phenotypic profiles. In contrast, the relatively genetically stable tumor microenvironment (TME) therefore provides an appealing therapeutic target, particularly since it plays an essential role in promoting cancer growth, immune tolerance, and acquired resistance to many therapies. Recently, a variety of different TME-targeted gene therapy and armed oncolytic strategies have been explored, with particular success observed in strategies targeting the cancer stroma, reducing tumor vasculature, and repolarizing the immunosuppressive microenvironment. Herein, we review the progress of these TME-targeting approaches and try to highlight those showing the greatest promise.

Olaparib increases the therapeutic index of hemithoracic irradiation compared with hemithoracic irradiation alone in a mouse lung cancer model.

BACKGROUND: The radiosensitising effect of the poly(ADP-ribose) polymerase inhibitor olaparib on tumours has been reported. However, its effect on normal tissues in combination with radiation has not been well studied. Herein, we investigated the therapeutic index of olaparib combined with hemithoracic radiation in a urethane-induced mouse lung cancer model. METHODS: To assess tolerability, A/J mice were treated with olaparib plus whole thorax radiation (13 Gy), body weight changes were monitored and normal tissue effects were assessed by histology. In anti-tumour (intervention) studies, A/J mice were injected with urethane to induce lung tumours, and were then treated with olaparib alone, left thorax radiation alone or the combination of olaparib plus left thorax radiation at 8 weeks (early intervention) or 18 weeks (late intervention) after urethane injection. Anti-tumour efficacy and normal tissue effects were assessed by visual inspection, magnetic resonance imaging and histology. RESULTS: Enhanced body weight loss and oesophageal toxicity were observed when olaparib was combined with whole thorax but not hemithorax radiation. In both the early and late intervention studies, olaparib increased the anti-tumour effects of hemithoracic irradiation without increasing lung toxicity. CONCLUSIONS: The addition of olaparib increased the therapeutic index of hemithoracic radiation in a mouse model of lung cancer.

Replication stress and chromatin context link ATM activation to a role in DNA replication.

ATM-mediated signaling in response to DNA damage is a barrier to tumorigenesis. Here we asked whether replication stress could also contribute to ATM signaling. We demonstrate that, in the absence of DNA damage, ATM responds to replication stress in a hypoxia-induced heterochromatin-like context. In certain hypoxic conditions, replication stress occurs in the absence of detectable DNA damage. Hypoxia also induces H3K9me3, a histone modification associated with gene repression and heterochromatin. Hypoxia-induced replication stress together with increased H3K9me3 leads to ATM activation. Importantly, ATM prevents the accumulation of DNA damage in hypoxia. Most significantly, we describe a stress-specific role for ATM in maintaining DNA replication rates in a background of increased H3K9me3. Furthermore, the ATM-mediated response to oncogene-induced replication stress is enhanced in hypoxic conditions. Together, these data indicate that hypoxia plays a critical role in the activation of the DNA damage response, therefore contributing to this barrier to tumorigenesis.

Overcoming acquired resistance to HSP90 inhibition by targeting JAK-STAT signalling in triple-negative breast cancer.

BACKGROUND: Due to the lack of effective therapies and poor prognosis in TNBC (triple-negative breast cancer) patients, there is a strong need to develop effective novel targeted therapies for this subtype of breast cancer. Inhibition of heat shock protein 90 (HSP90), a conserved molecular chaperone that is involved in the regulation of oncogenic client proteins, has shown to be a promising therapeutic approach for TNBC. However, both intrinsic and acquired resistance to HSP90 inhibitors (HSP90i) limits their effectiveness in cancer patients. METHODS: We developed models of acquired resistance to HSP90i by prolonged exposure of TNBC cells to HSP90i (ganetespib) in vitro. Whole transcriptome profiling and a 328-compound bioactive small molecule screen were performed on these cells to identify the molecular basis of acquired resistance to HSP90i and potential therapeutic approaches to overcome resistance. RESULTS: Among a panel of seven TNBC cell lines, the most sensitive cell line (Hs578T) to HSP90i was selected as an in vitro model to investigate acquired resistance to HSP90i. Two independent HSP90i-resistant clones were successfully isolated which both showed absence of client proteins degradation, apoptosis induction and G2/M cell cycle arrest after treatment with HSP90i. Gene expression profiling and pathway enrichment analysis demonstrate significant activation of the survival JAK-STAT signalling pathway in both HSP90i-resistant clones, possibly through IL6 autocrine signalling. A bioactive small molecule screen also demonstrated that the HSP90i-resistant clones showed selective sensitivity to JAK2 inhibition. Inhibition of JAK and HSP90 caused higher induction of apoptosis, despite prior acquired resistance to HSP90i. CONCLUSIONS: Acquired resistance to HSP90i in TNBC cells is associated with an upregulated JAK-STAT signalling pathway. A combined inhibition of the JAK-STAT signalling pathway and HSP90 could overcome this resistance. The benefits of the combined therapy could be explored further for the development of effective targeted therapy in TNBC patients.

TOPK modulates tumour-specific radiosensitivity and correlates with recurrence after prostate radiotherapy.

BACKGROUND: Tumour-specific radiosensitising treatments may enhance the efficacy of radiotherapy without exacerbating side effects. In this study we determined the radiation response following depletion or inhibition of TOPK, a mitogen-activated protein kinase kinase family Ser/Thr protein kinase that is upregulated in many cancers. METHODS: Radiation response was studied in a wide range of cancer cell lines and normal cells using colony formation assays. The effect on cell cycle progression was assessed and the relationship between TOPK expression and therapeutic efficacy was studied in a cohort of 128 prostate cancer patients treated with radical radiotherapy. RESULTS: TOPK knockdown did not alter radiation response in normal tissues, but significantly enhanced radiosensitivity in cancer cells. This result was recapitulated in TOPK knockout cells and with the TOPK inhibitor, OTS964. TOPK depletion altered the G1/S transition and G2/M arrest in response to radiation. Furthermore, TOPK depletion increased chromosomal aberrations, multinucleation and apoptotic cell death after irradiation. These results suggest a possible role for TOPK in the radiation-induced DNA damage checkpoints. These findings have clinical relevance, as elevated TOPK protein expression was associated with poorer clinical outcomes in prostate cancer patients treated with radical radiotherapy. CONCLUSIONS: This study demonstrates that TOPK disruption may cause tumour-specific radiosensitisation in multiple different tumour types.

Orthotopic and metastatic tumour models in preclinical cancer research.

Mouse models of disease play a pivotal role at all stages of cancer drug development. Cell-line derived subcutaneous tumour models are predominant in early drug discovery, but there is growing recognition of the importance of the more complex orthotopic and metastatic tumour models for understanding both target biology in the correct tissue context, and the impact of the tumour microenvironment and the immune system in responses to treatment. The aim of this review is to highlight the value that orthotopic and metastatic models bring to the study of tumour biology and drug development while pointing out those models that are most likely to be encountered in the literature. Important developments in orthotopic models, such as the increasing use of early passage patient material (PDXs, organoids) and humanised mouse models are discussed, as these approaches have the potential to increase the predictive value of preclinical studies, and ultimately improve the success rate of anticancer drugs in clinical trials.

Evaluation of novel combined carbogen USPIO (CUSPIO) imaging biomarkers in assessing the antiangiogenic effects of cediranib (AZD2171) in rat C6 gliomas.

The recently described combined carbogen USPIO (CUSPIO) magnetic resonance imaging (MRI) method uses spatial correlations in independent imaging biomarkers to assess specific components of tumor vascular structure and function. Our study aimed to evaluate CUSPIO biomarkers for the assessment of tumor response to antiangiogenic therapy. CUSPIO imaging was performed in subcutaneous rat C6 gliomas before and 2 days after treatment with the potent VEGF-signaling inhibitor cediranib (n = 12), or vehicle (n = 12). Histological validation of Hoechst 33342 uptake (perfusion), smooth muscle actin staining (maturation), pimonidazole adduct formation (hypoxia) and necrosis were sought. Following treatment, there was a significant decrease in fractional blood volume (-43%, p < 0.01) and a significant increase in hemodynamic vascular functionality (treatment altered ΔR(2) *(carbogen) from 1.2 to -0.2 s(-1) , p < 0.05). CUSPIO imaging revealed an overall significant decrease in plasma perfusion (-27%, p < 0.05) following cediranib treatment, that was associated with selective effects on immature blood vessels. The CUSPIO responses were associated with a significant 15% reduction in Hoechst 33342 uptake (p < 0.05), but no significant difference in vascular maturation or necrosis. Additionally, treatment with cediranib resulted in a significant 40% increase in tumor hypoxia (p < 0.05). The CUSPIO imaging method provides novel and more specific biomarkers of tumor vessel maturity and vascular hemodynamics, and their response to VEGF-signaling inhibition, compared to current MR imaging biomarkers utilized in the clinic. Such biomarkers may prove effective in longitudinally monitoring tumor vascular remodeling and/or evasive resistance in response to antiangiogenic therapy.

The cell-line-derived subcutaneous tumor model in preclinical cancer research.

Tumor-bearing experimental animals are essential for preclinical cancer drug development. A broad range of tumor models is available, with the simplest and most widely used involving a tumor of mouse or human origin growing beneath the skin of a mouse: the subcutaneous tumor model. Here, we outline the different types of in vivo tumor model, including some of their advantages and disadvantages and how they fit into the drug-development process. We then describe in more detail the subcutaneous tumor model and key steps needed to establish it in the laboratory, namely: choosing the mouse strain and tumor cells; cell culture, preparation and injection of tumor cells; determining tumor volume; mouse welfare; and an appropriate experimental end point. The protocol leads to subcutaneous tumor growth usually within 1-3 weeks of cell injection and is suitable for those with experience in tissue culture and mouse experimentation.

CHK1 inhibition exacerbates replication stress induced by IGF blockade.

We recently reported that genetic or pharmacological inhibition of insulin-like growth factor receptor (IGF-1R) slows DNA replication and induces replication stress by downregulating the regulatory subunit RRM2 of ribonucleotide reductase, perturbing deoxynucleotide triphosphate (dNTP) supply. Aiming to exploit this effect in therapy we performed a compound screen in five breast cancer cell lines with IGF neutralising antibody xentuzumab. Inhibitor of checkpoint kinase CHK1 was identified as a top screen hit. Co-inhibition of IGF and CHK1 caused synergistic suppression of cell viability, cell survival and tumour growth in 2D cell culture, 3D spheroid cultures and in vivo. Investigating the mechanism of synthetic lethality, we reveal that CHK1 inhibition in IGF-1R depleted or inhibited cells further downregulated RRM2, reduced dNTP supply and profoundly delayed replication fork progression. These effects resulted in significant accumulation of unreplicated single-stranded DNA and increased cell death, indicative of replication catastrophe. Similar phenotypes were induced by IGF:WEE1 co-inhibition, also via exacerbation of RRM2 downregulation. Exogenous RRM2 expression rescued hallmarks of replication stress induced by co-inhibiting IGF with CHK1 or WEE1, identifying RRM2 as a critical target of the functional IGF:CHK1 and IGF:WEE1 interactions. These data identify novel therapeutic vulnerabilities and may inform future trials of IGF inhibitory drugs.

Investigating temporal fluctuations in tumor vasculature with combined carbogen and ultrasmall superparamagnetic iron oxide particle (CUSPIO) imaging.

A combined carbogen ultrasmall superparamagnetic iron oxide (USPIO) imaging protocol was developed and applied in vivo in two murine colorectal tumor xenograft models, HCT116 and SW1222, with established disparate vascular morphology, to investigate whether additional information could be extracted from the combination of two susceptibility MRI biomarkers. Tumors were imaged before and during carbogen breathing and subsequently following intravenous administration of USPIO particles. A novel segmentation method was applied to the image data, from which six categories of R(2)* response were identified, and compared with histological analysis of the vasculature. In particular, a strong association between a negative ΔR(2)*(carbogen) followed by positive ΔR(2)*(USPIO) with the uptake of the perfusion marker Hoechst 33342 was determined. Regions of tumor tissue where there was a significant ΔR(2)*(carbogen) but no significant ΔR(2)*(USPIO) were also identified, suggesting these regions became temporally isolated from the vascular supply during the experimental timecourse. These areas correlated with regions of tumor tissue where there was CD31 staining but no Hoechst 33342 uptake. Significantly, different combined carbogen USPIO responses were determined between the two tumor models. Combining ΔR(2)*(carbogen) and ΔR(2)*(USPIO) with a novel segmentation scheme can facilitate the interpretation of susceptibility contrast MRI data and enable a deeper interrogation of tumor vascular function and architecture.

DNAPK Inhibition Preferentially Compromises the Repair of Radiation-induced DNA Double-strand Breaks in Chronically Hypoxic Tumor Cells in Xenograft Models.

Radiation-induced DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) and nonhomologous end joining (NHEJ). Recently, it has been found that chronic tumor hypoxia compromises HR repair of DNA DSBs but activates the NHEJ protein DNAPK. We therefore hypothesized that inhibition of DNAPK can preferentially potentiate the sensitivity of chronically hypoxic cancer cells to radiation through contextual synthetic lethality in vivo In this study, we investigated the impact of DNAPK inhibition by a novel selective DNAPK inhibitor, NU5455, on the repair of radiation-induced DNA DSBs in chronically hypoxic and nonhypoxic cells across a range of xenograft models. We found that NU5455 inhibited DSB repair following radiation in both chronically hypoxic and nonhypoxic tumor cells. Most importantly, the inhibitory effect was more pronounced in chronically hypoxic tumor cells than in nonhypoxic tumor cells. This is the first in vivo study to indicate that DNAPK inhibition may preferentially sensitize chronically hypoxic tumor cells to radiotherapy, suggesting a broader therapeutic window for transient DNAPK inhibition combined with radiotherapy.

Targeting IGF Perturbs Global Replication through Ribonucleotide Reductase Dysfunction.

Inhibition of IGF receptor (IGF1R) delays repair of radiation-induced DNA double-strand breaks (DSB), prompting us to investigate whether IGF1R influences endogenous DNA damage. Here we demonstrate that IGF1R inhibition generates endogenous DNA lesions protected by 53BP1 bodies, indicating under-replicated DNA. In cancer cells, inhibition or depletion of IGF1R delayed replication fork progression accompanied by activation of ATR-CHK1 signaling and the intra-S-phase checkpoint. This phenotype reflected unanticipated regulation of global replication by IGF1 mediated via AKT, MEK/ERK, and JUN to influence expression of ribonucleotide reductase (RNR) subunit RRM2. Consequently, inhibition or depletion of IGF1R downregulated RRM2, compromising RNR function and perturbing dNTP supply. The resulting delay in fork progression and hallmarks of replication stress were rescued by RRM2 overexpression, confirming RRM2 as the critical factor through which IGF1 regulates replication. Suspecting existence of a backup pathway protecting from toxic sequelae of replication stress, targeted compound screens in breast cancer cells identified synergy between IGF inhibition and ATM loss. Reciprocal screens of ATM-proficient/deficient fibroblasts identified an IGF1R inhibitor as the top hit. IGF inhibition selectively compromised growth of ATM-null cells and spheroids and caused regression of ATM-null xenografts. This synthetic-lethal effect reflected conversion of single-stranded lesions in IGF-inhibited cells into toxic DSBs upon ATM inhibition. Overall, these data implicate IGF1R in alleviating replication stress, and the reciprocal IGF:ATM codependence we identify provides an approach to exploit this effect in ATM-deficient cancers. SIGNIFICANCE: This study identifies regulation of ribonucleotide reductase function and dNTP supply by IGFs and demonstrates that IGF axis blockade induces replication stress and reciprocal codependence on ATM. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2128/F1.large.jpg.

Germline and Somatic Genetic Variants in the p53 Pathway Interact to Affect Cancer Risk, Progression, and Drug Response.

Insights into oncogenesis derived from cancer susceptibility loci (SNP) hold the potential to facilitate better cancer management and treatment through precision oncology. However, therapeutic insights have thus far been limited by our current lack of understanding regarding both interactions of these loci with somatic cancer driver mutations and their influence on tumorigenesis. For example, although both germline and somatic genetic variation to the p53 tumor suppressor pathway are known to promote tumorigenesis, little is known about the extent to which such variants cooperate to alter pathway activity. Here we hypothesize that cancer risk-associated germline variants interact with somatic TP53 mutational status to modify cancer risk, progression, and response to therapy. Focusing on a cancer risk SNP (rs78378222) with a well-documented ability to directly influence p53 activity as well as integration of germline datasets relating to cancer susceptibility with tumor data capturing somatically-acquired genetic variation provided supportive evidence for this hypothesis. Integration of germline and somatic genetic data enabled identification of a novel entry point for therapeutic manipulation of p53 activities. A cluster of cancer risk SNPs resulted in increased expression of prosurvival p53 target gene KITLG and attenuation of p53-mediated responses to genotoxic therapies, which were reversed by pharmacologic inhibition of the prosurvival c-KIT signal. Together, our results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and identify novel combinatorial therapies. SIGNIFICANCE: These results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and present novel therapeutic targets.

Baseline vascular endothelial growth factor concentration as a potential predictive marker of benefit from vandetanib in non-small cell lung cancer.

PURPOSE: Vandetanib [vascular endothelial growth factor (VEGF) receptor/epidermal growth factor receptor/RET inhibitor] has shown improvements in progression-free survival (PFS) in advanced non-small cell lung cancer in three randomized phase II studies: vandetanib versus gefitinib (study 3), docetaxel +/- vandetanib (study 6), and carboplatin-paclitaxel and/or vandetanib (study 7). In study 7, vandetanib monotherapy was inferior to carboplatin-paclitaxel. We performed an exploratory retrospective analysis of the relationship between baseline circulating VEGF concentrations and PFS. EXPERIMENTAL DESIGN: Mean baseline VEGF levels were determined by ELISA from two baseline samples of plasma (163 of 168 patients, study 3; 65 of 127, study 6) or serum (144 of 181, study 7). High baseline VEGF values were above the immunoassay reference range for healthy subjects; low baseline VEGF values were within the range. RESULTS: Patients with low baseline VEGF had a lower risk of disease progression with vandetanib versus gefitinib [hazard ratio (HR), 0.55; 95% confidence interval (95% CI), 0.35-0.86; P = 0.01] or vandetanib 100 mg/d + docetaxel versus docetaxel (HR, 0.25; 95% CI, 0.09-0.68; P = 0.01). High VEGF patients had a similar risk of disease progression with vandetanib monotherapy versus gefitinib (HR, 1.03; 95% CI, 0.60-1.75; P = 0.92) or vandetanib 100 mg/d + docetaxel versus docetaxel (HR, 0.95; 95% CI, 0.25-3.61; P = 0.94). In study 7, low VEGF patients had a similar risk of disease progression with vandetanib monotherapy 300 mg/d versus carboplatin-paclitaxel (HR, 0.80; 95% CI, 0.41-1.56; P = 0.51); high VEGF patients progressed more quickly (HR, 1.60; 95% CI, 0.81-3.15; P = 0.17). CONCLUSIONS: These analyses suggest that low baseline circulating VEGF may be predictive of PFS advantage in patients with advanced non-small cell lung cancer receiving vandetanib versus gefitinib or vandetanib + docetaxel versus docetaxel. Moreover, patients with low VEGF levels may have a similar outcome with either vandetanib monotherapy or carboplatin-paclitaxel.

Selective DNA-PKcs inhibition extends the therapeutic index of localized radiotherapy and chemotherapy.

Potentiating radiotherapy and chemotherapy by inhibiting DNA damage repair is proposed as a therapeutic strategy to improve outcomes for patients with solid tumors. However, this approach risks enhancing normal tissue toxicity as much as tumor toxicity, thereby limiting its translational impact. Using NU5455, a newly identified highly selective oral inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity, we found that it was indeed possible to preferentially augment the effect of targeted radiotherapy on human orthotopic lung tumors without influencing acute DNA damage or a late radiation-induced toxicity (fibrosis) to normal mouse lung. Furthermore, while NU5455 administration increased both the efficacy and the toxicity of a parenterally administered topoisomerase inhibitor, it enhanced the activity of doxorubicin released locally in liver tumor xenografts without inducing any adverse effect. This strategy is particularly relevant to hepatocellular cancer, which is treated clinically with localized drug-eluting beads and for which DNA-PKcs activity is reported to confer resistance to treatment. We conclude that transient pharmacological inhibition of DNA-PKcs activity is effective and tolerable when combined with localized DNA-damaging therapies and thus has promising clinical potential.

CONCORDE: A phase I platform study of novel agents in combination with conventional radiotherapy in non-small-cell lung cancer.

Lung cancer is the leading cause of cancer mortality worldwide and most patients are unsuitable for 'gold standard' treatment, which is concurrent chemoradiotherapy. CONCORDE is a platform study seeking to establish the toxicity profiles of multiple novel radiosensitisers targeting DNA repair proteins in patients treated with sequential chemoradiotherapy. Time-to-event continual reassessment will facilitate efficient dose-finding.

Identification of tyrosine 806 as a molecular determinant of RET kinase sensitivity to ZD6474.

ZD6474 (vandetanib, Zactima, Astra Zeneca) is an anilinoquinazoline used to target the receptor tyrosine kinase RET in familial and sporadic thyroid carcinoma (IC(50): 100 nM). The aim of this study was to identify molecular determinants of RET sensitivity to ZD6474. Here, we show that mutation of RET tyrosine 806 to cysteine (Y806C) induced RET kinase resistance to ZD6474 (IC(50): 933 nM). Y806 maps close to the gate-keeper position at the RET kinase nucleotide-binding pocket. Although tyrosine 806 is a RET auto-phosphorylation site, its substitution to phenylalanine (Y806F) did not markedly affect RET susceptibility to ZD6474 (IC(50): 87 nM), suggesting that phosphorylation of Y806 is not required for compound binding. Accordingly, the introduction of a phosphomimetic residue (Y806E) also caused resistance to ZD6474, albeit of a lesser degree (IC(50): 512 nM) than the cysteine mutation. Y806C/E RET mutants were also resistant to ZD6474 with respect to intracellular signalling and activation of an AP1-responsive promoter. We conclude that Y806 is a molecular determinant of RET sensitivity to ZD6474. Y806C is a natural RET mutation identified in a patient affected by multiple endocrine neoplasia type 2B. Based on its rare occurrence, it is unlikely that Y806C will be a frequent cause of refractoriness to ZD6474; however, it may be envisaged that mutations at this site can mediate secondary resistance formation in patients treated with the compound.

Vandetanib inhibits growth of adenoid cystic carcinoma in an orthotopic nude mouse model.

PURPOSE: Adenoid cystic carcinoma (ACC) can often be controlled with surgery and postoperative adjuvant radiotherapy but is also characterized by late local recurrence and distant metastasis. No effective systemic therapeutic agents have been found to alter the natural history of ACC. Therefore, new therapeutic approaches are needed. In this study, we evaluated whether vandetanib (Zactima), a potent inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) and epidermal growth factor receptor (EGFR) tyrosine kinases, had antitumor efficacy in vitro and in an orthotopic nude mouse model of human ACC. EXPERIMENTAL DESIGN: The in vitro effects of vandetanib were assessed in three ACC cell lines on cell growth, apoptosis, and VEGFR-2 and EGFR phosphorylation levels. The in vivo antitumor activity of vandetanib was examined in nude mice bearing parotid gland ACC tumors. The mice were treated for 4 weeks with vandetanib (50 mg/kg/d) or placebo (control). Tumors were resected at necropsy, and immunohistochemical and immunofluorescence staining were done. RESULTS: In vitro, vandetanib caused dose-dependent inhibition of VEGFR-2 and EGFR phosphorylation in ACC cells. Vandetanib also inhibited the cell proliferation and induced their dose-dependent apoptosis. In vivo, mice in the vandetanib group had tumor volumes significantly lower than those in the control group (P < 0.01). In addition, immunohistochemical staining showed a decrease in microvessel density and an increase in apoptosis of both tumor cells and endothelial cells within the tumor xenografts. CONCLUSION: These results suggest that vandetanib inhibits the growth of ACC in vitro and in vivo, making it a promising novel agent for the treatment of ACC.

Dual inhibition of VEGFR and EGFR signaling reduces the incidence and size of intestinal adenomas in Apc(Min/+) mice.

Both the epidermal growth factor (EGF) and the vascular endothelial growth factor (VEGF) pathways are associated with intestinal cancer, and therapeutic approaches targeting either EGF receptor (EGFR) or VEGF receptor (VEGFR) signaling have recently been approved for patients with advanced colorectal cancer. The Apc(Min/+) mouse is a well-characterized in vivo model of intestinal tumorigenesis, and animals with this genetic mutation develop macroscopically detectable adenomas from approximately 6 weeks of age. Previous work in the Apc(Min/+) mouse has shown that therapeutic approaches targeting either VEGFR or EGFR signaling affect predominantly the size or number of adenomas, respectively. In this study, we have assessed the effect of inhibiting both these key pathways simultaneously using ZD6474 (Vandetanib, ZACTIMA), a selective inhibitor of VEGFR and EGFR tyrosine kinases. To assess the effects of ZD6474 on early- and later-stage disease, treatment was initiated in 6- and 10-week-old Apc(Min/+) mice for 28 days. ZD6474 markedly reduced both the number and the size of polyps when administered at either an early or a later stage of polyp development. This reduction in both adenoma number and size resulted in a total reduction in tumor burden in the small intestine of nearly 75% in both studies (P < 0.01). The current data build on the concept that EGFR-dependent tumor cell proliferation and VEGF/VEGFR2-dependent angiogenesis and survival are distinct key mechanisms in polyp development. Pharmacologic inhibition of both signaling pathways has significant antitumor effects at both early and late stages of polyp development. Therefore, targeting both VEGFR- and EGFR-dependent signaling may be a beneficial strategy in early intestinal cancer.