Hypoxia Potentiates the Radiation-Sensitizing Effect of Olaparib in Human Non-Small Cell Lung Cancer Xenografts by Contextual Synthetic Lethality.

PURPOSE: Poly(ADP-ribose) polymerase (PARP) inhibitors potentiate radiation therapy in preclinical models of human non-small cell lung cancer (NSCLC) and other types of cancer. However, the mechanisms underlying radiosensitization in vivo are incompletely understood. Herein, we investigated the impact of hypoxia on radiosensitization by the PARP inhibitor olaparib in human NSCLC xenograft models. METHODS AND MATERIALS: NSCLC Calu-6 and Calu-3 cells were irradiated in the presence of olaparib or vehicle under normoxic (21% O2) or hypoxic (1% O2) conditions. In vitro radiosensitivity was assessed by clonogenic survival assay and γH2AX foci assay. Established Calu-6 and Calu-3 subcutaneous xenografts were treated with olaparib (50 mg/kg, daily for 3 days), radiation (10 Gy), or both. Tumors (n=3/group) were collected 24 or 72 hours after the first treatment. Immunohistochemistry was performed to assess hypoxia (carbonic anhydrase IX [CA9]), vessels (CD31), DNA double strand breaks (DSB) (γH2AX), and apoptosis (cleaved caspase 3 [CC3]). The remaining xenografts (n=6/group) were monitored for tumor growth. RESULTS: In vitro, olaparib showed a greater radiation-sensitizing effect in Calu-3 and Calu-6 cells in hypoxic conditions (1% O2). In vivo, Calu-3 tumors were well-oxygenated, whereas Calu-6 tumors had extensive regions of hypoxia associated with down-regulation of the homologous recombination protein RAD51. Olaparib treatment increased unrepaired DNA DSB (P<.001) and apoptosis (P<.001) in hypoxic cells of Calu-6 tumors following radiation, whereas it had no significant effect on radiation-induced DNA damage response in nonhypoxic cells of Calu-6 tumors or in the tumor cells of well-oxygenated Calu-3 tumors. Consequently, olaparib significantly increased radiation-induced growth inhibition in Calu-6 tumors (P<.001) but not in Calu-3 tumors. CONCLUSIONS: Our data suggest that hypoxia potentiates the radiation-sensitizing effects of olaparib by contextual synthetic killing, and that tumor hypoxia may be a potential biomarker for selecting patients who may get the greatest benefit from the addition of olaparib to radiation therapy.

Acute vascular response to cediranib treatment in human non-small-cell lung cancer xenografts with different tumour stromal architecture.

OBJECTIVES: Tumours can be categorised based on their stromal architecture into tumour vessel and stromal vessel phenotypes, and the phenotypes have been suggested to define tumour response to chronic treatment with a VEGFR2 antibody. However, it is unclear whether the vascular phenotypes of tumours associate with acute vascular response to VEGFR tyrosine kinase inhibitors (TKI), or whether the early changes in vascular function are associated with subsequent changes in tumour size. This study was sought to address these questions by using xenograft models of human non-small cell lung cancer (NSCLC) representing stromal vessel phenotype (Calu-3) and tumour vessel phenotype (Calu-6), respectively. METHODS: For dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), nude mice bearing established Calu-3 or Calu-6 xenografts were treated with a potent pan-VEGFR TKI, cediranib (6 mg/kg), at 0 h and 22 h. DCE-MRI was performed 2h before the first dose and 2h after the second dose of cediranib to examine acute changes in tumour vessel perfusion. Tumours were harvested for hypoxia detection by CA9 immunohistochemistry. For tumour growth study, mice carrying established Calu-3 or Calu-6 tumours were treated with cediranib once daily for 5 days. RESULTS: Twenty-four hours after cediranib administration, the perfusion of Calu-3 tumours was markedly reduced, with a significant increase in hypoxia. In contrast, neither perfusion nor hypoxia was significantly affected in Calu-6 tumours. Tumour regressions were induced in Calu-3 xenografts, but not in Calu-6 xenografts, although there was a trend towards tumour growth inhibition after 5 days of cediranib treatment. CONCLUSION: These findings suggest that tumour stromal architecture may associate with acute tumour vascular response to VEGFR TKI, and this acute tumour vascular response may be a promising early predictive marker of response to VEGFR TKI in NSCLC.

Vascular endothelial growth factor directly stimulates tumour cell proliferation in non-small cell lung cancer.

Vascular endothelial growth factor (VEGF) is a key stimulator of physiological and pathological angiogenesis. VEGF signals primarily through VEGF receptor 2 (VEGFR2), a receptor tyrosine kinase whose expression is found predominantly on endothelial cells. The purpose of this study was to determine the role of VEGFR2 expression in NSCLC cells. NSCLC cells and tissue sections were stained for VEGFR2 expression by immunohistochemistry (IHC). Immunoblotting and ELISA were used to determine the activation and inhibition of VEGFR2 and its downstream signalling pathways. Five-day proliferation assays were carried out in the presence or absence of VEGF. IHC analysis of NSCLC demonstrated tumour cell VEGFR2 expression in 20% of samples. Immunoblot analysis showed expression of VEGFR2 protein in 3/8 NSCLC cell lines that correlated with VEGFR2 mRNA expression levels. VEGF-dependent VEGFR2 activation was apparent in NSCLC cells, and was associated with increased tumor cell proliferation. Cediranib treatment or siRNA against VEGFR2 inhibited VEGF-dependent increases in cell proliferation. Inhibition of VEGFR2 tyrosine kinase activity using cediranib was more effective than inhibition of AKT (MK2206) or MEK (AZD6244) for overcoming VEGFR2-driven cell proliferation. VEGF treatment did not affect cell survival following treatment with radiation, cisplatin, docetaxel or gemcitabine. Our data suggest that a subset of NSCLC tumour cells express functional VEGFR2 which can act to promote VEGF-dependent tumour cell growth. In this tumour subset, therapies targeting VEGFR2 signalling, such as cediranib, have the potential to inhibit both tumour cell proliferation and angiogenesis.

Combining AKT inhibition with chloroquine and gefitinib prevents compensatory autophagy and induces cell death in EGFR mutated NSCLC cells.

Although non-small cell lung cancer (NSCLC) patients with EGFR mutation positive (EGFR M+) tumors initially respond well to EGFR tyrosine kinase inhibitor (TKI) monotherapy, the responses are usually incomplete. In this study we show that AKT inhibition, most importantly AKT2 inhibition, synergises with EGFR TKI inhibition to increase cell killing in EGFR M+ NSCLC cells. However, our data also suggest that the synergistic pro-apoptotic effects may be stunted due to a prosurvival autophagy response induced by AKT inhibition. Consequently, inhibiting autophagy with chloroquine significantly enhanced tumor cell death induced by gefitinib and AKT inhibitors in EGFR M+ cells in vitro, and produced greater tumor shrinkage in EGFR M+ xenografts in vivo. Together, our findings suggest that adding chloroquine to EGFR and AKT inhibition has the potential to improve tumor responses in EGFR M+ NSCLC, and that selective targeting of AKT2 may provide a new treatment option in NSCLC.

Short-course treatment with gefitinib enhances curative potential of radiation therapy in a mouse model of human non-small cell lung cancer.

PURPOSE: To evaluate the combination of radiation and an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) in preclinical models of human non-small cell lung cancer. METHODS AND MATERIALS: Sensitivity to an EGFR TKI (gefitinib) or radiation was assessed using proliferation assays and clonogenic survival assays. Effects on receptor signal transduction pathways (pEGFR, pAKT, pMAPK) and apoptosis (percentage of cleaved PARP Poly (ADP-ribose) polymerase (PARP)) were assessed by Western blotting. Radiation-induced DNA damage was assessed by γH2AX immunofluorescence. Established (≥ 100 mm(3)) EGFR-mutated (HCC287) or EGFR wild-type (A549) subcutaneous xenografts were treated with radiation (10 Gy, day 1) or gefitinib (50 mg/kg, orally, on days 1-3) or both. RESULTS: In non-small cell lung cancer (NSCLC) cell lines with activating EGFR mutations (PC9 or HCC827), gefitinib treatment markedly reduced pEGFR, pAKT, and pMAPK levels and was associated with an increase in cleaved PARP but not in γH2AX foci. Radiation treatment increased the mean number of γH2AX foci per cell but did not significantly affect EGFR signaling. In contrast, NSCLC cell lines with EGFR T790M (H1975) or wild-type EGFR (A549) were insensitive to gefitinib treatment. The combination of gefitinib and radiation treatment in cell culture produced additive cell killing with no evidence of synergy. In xenograft models, a short course of gefitinib (3 days) did not significantly increase the activity of radiation treatment in wild-type EGFR (A549) tumors (P=.27), whereas this combination markedly increased the activity of radiation (P<.001) or gefitinib alone (P=.002) in EGFR-mutated HCC827 tumors, producing sustained tumor regressions. CONCLUSIONS: Gefitinib treatment increases clonogenic cell killing by radiation but only in cell lines sensitive to gefitinib alone. Our data suggest additive rather than synergistic interactions between gefitinib and radiation and that a combination of short-course gefitinib and high-dose/-fraction radiation may have the greatest potential against the subsets of lung cancers harboring activating mutations in the EGFR gene.

Growth and differentiation factor 9 (GDF-9) induces epithelial-mesenchymal transition in prostate cancer cells.

The role of bone morphogenetic proteins in the progression and metastasis of prostate cancer is a topic that has undergone extensive research. This study investigates the role of BMP member growth and differentiation factor 9 (GDF-9) in the progression of this disease. GDF-9 was over-expressed and knocked-down in PC-3 cells, respectively. Furthermore, along with the use of a generated recombinant GDF-9 protein, these cells were then analyzed for any changes in their invasiveness and expression of epithelial-mesenchymal transition (EMT) associated genes. GDF-9 was shown to promote the invasiveness of PC-3 cells together with an induction in the expression of genes including SNAI1, RhoC, ROCK-1, and N-cadherin, while reducing levels of E-cadherin. These expression changes are characteristic of the onset of EMT, and resulted in the cells having a more mesenchymal-like morphology. Treating these cells with activin-like kinase-5 (ALK-5) inhibitor, demonstrated that GDF-9 induced up-regulation of these molecules was ALK-5 dependant. This study shows that in PC-3 cells, GDF-9 signaling via ALK-5, can promote cell invasiveness via a complex network of signaling molecules that work together to trigger the process of EMT, and thereby aid in the aggressiveness and progression of prostate cancer cells.

Growth and differentiation factor-9 promotes adhesive and motile capacity of prostate cancer cells by up-regulating FAK and Paxillin via Smad dependent pathway.

The majority of advanced prostate cancers metastasis to the bone. Mediators of bone remodelling, the bone morphogenetic proteins have extensively been implicated in the progression and metastasis of prostate cancer. The present study investigated the function of BMP member GDF-9, in prostate cancer. We overexpressed GDF9 in PC-3 cells using a mammalian expression construct, and knocked-down with the use of ribozyme transgenes. These cells were further used in in vitro adhesion and motility assays, in order to determine the effect of GDF-9 on these properties. Recombinant GDF-9 was generated to treat PC-3 WT cells before further analysing the effect on adhesion. The GDF-9 overexpressing PC-3 cells demonstrated a significantly enhanced adhesive and motile capacity compared to their controls. The opposite effect was seen in the GDF-9 knock-down cells. In addition, treating PC-3 cells with rh-GDF-9 resulted in them becoming more adhesive. Both endogenous and exogenous GDF-9 was demonstrated to up-regulate focal adhesion associated proteins FAK and paxillin which contribute to promoted cell adhesion and motility. With the use of a Smad3 inhibitor, this effect was inhibited suggesting that GDF-9 signals via Smad3 to up-regulate expression of these proteins. This study shows that GDF-9 can promote the motile and adhesive capacity of PC-3 prostate cancer cells by up-regulating expression of FAK and paxillin in a Smad dependent manner, suggesting a pro-tumourigenic role for GDF-9 in prostate cancer.

GDF-9 promotes the growth of prostate cancer cells by protecting them from apoptosis.

Bone morphogenetic proteins (BMPs) have long been implicated in the process of prostate cancer progression and bone metastasis. This current study investigates the role of GDF-9, a BMP member, in prostate cancer. GDF-9 was over-expressed in PC-3 cells using a mammalian expression construct. Additionally, GDF-9 ribozyme transgenes were generated in order to knock down the expression of GDF-9 in PC-3 and DU-145 cells. These cells were then used in in vitro growth assays in order to determine the effect of GDF-9 on prostate cancer cell growth. Recombinant GDF-9 was also generated and used to treat both cell lines before carrying out further growth assays. Levels of apoptosis were subsequently analyzed using flow cytometry. Cell growth was significantly increased in the GDF-9 over-expressing cells compared to the two controls. The cell growth rate at day 5 was significantly greater in the PC-3(GDF-9exp.) (1,131.1 +/- 79.1%) compared to both PC-3(WT) (563.9 +/- 90.6%) and PC-3(pEF) (763.3 +/- 82.0%), P

Bone morphogenetic proteins in development and progression of breast cancer and therapeutic potential (review).

Bone morphogenetic proteins (BMPs) belong to the TGF-beta superfamily, which plays important roles in foetal and postnatal development and also maintains the homeostasis of various tissues and organs. Due to the critical role played by BMPs in bone formation and bone turnover, the implication of these molecules in bone metastasis has been intensively studied over the past decade. BMPs have been implicated in the development and progression of solid tumours, particularly the disease-specific bone metastasis. In breast cancer, a tumour type which most commonly metastasizes to bones, aberrations of both BMP expression and their signalling have been recently demonstrated. These aberrations have certain correlations with the development and progression of the disease. Recent in vitro studies have also demonstrated that BMPs can regulate a range of biological functions of breast cancer cells. Targeting BMPs or BMP signalling may provide novel therapeutic approaches for breast cancer. In the current review, we discuss the present knowledge on BMP abnormalities and their implication in the development and progression of breast cancer, particularly in the disease-specific bone metastasis.

Reduced expression of BMPR-IB correlates with poor prognosis and increased proliferation of breast cancer cells.

BACKGROUND: Breast cancer is the leading female cancer in the UK. Recent work has shown that Bone Morphogenetic Proteins (BMPs) and their receptors may be involved in the progression of breast cancer. The aim of the current study is to identify the role of BMPR-IB, one of these receptors, in breast cancer. MATERIALS AND METHODS: Expression of BMPR-IB was examined in a cohort of breast tissue samples. The transcript level was determined using quantitative real time-PCR and protein levels were assessed with immunohistochemical staining (IHC). Constructed ribozyme transgenes were used to knock-down BMPR-IB in MDA-MB-231 cells, and the effect this had on in vitro cell growth was examined. RESULTS: Decreased staining of BMPR-IB was seen in most of the breast tumour samples examined compared to normal breast tissues. Q-PCR analysis revealed lower expression levels of BMPR-IB transcript in patient samples with a predicted poor prognosis (Nottingham prognosis index (NPI) >5.4) (8.2+/-17, p=0.03) compared to samples with a good prognosis (NPI <3.4) (469+/-244). Higher BMPR-IB expression was seen in samples from disease free patients compared to those with poorer prognosis; including patients with metastasis, local recurrence and breast cancer deaths, (p=0.026). Furthermore, down-regulation of BMPR-IB in MDA-MB-231cells led to a promotion of in vitro cell growth. CONCLUSION: This study shows that decreased expression of BMPR-IB correlates with poor prognosis in breast cancer patients and leads to increased cell proliferation of breast cancer cells in vitro. This suggests that BMPR-IB mediates an inhibitory effect on breast cancer cells.